Polypeptides having oxaloacetate hydrolase activity and nucleic acids encoding same

ABSTRACT

The present invention relates to isolated polypeptides having oxaloacetate hydrolase activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods for producing and using the polypeptides.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims the benefit of U.S. Provisional Application No.60/493,708, filed Aug. 8, 2003, which application is incorporated hereinby reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to isolated polypeptides havingoxaloacetate hydrolase activity and isolated polynucleotides encodingthe polypeptides. The invention also relates to nucleic acid constructs,vectors, and host cells comprising the polynucleotides as well asmethods for producing and using the polypeptides.

2. Description of the Related Art

Lignin is an aromatic polymer occurring in the woody tissue of higherplants. Due to its hydrophobicity and complex random structure lackingregular hydrolyzable bonds, lignin is poorly degraded by most organisms.The best degraders of lignin are white rot fungi that produceextracellular peroxidases and laccases, which are involved in theinitial attack of lignin.

Manganese-dependent peroxidase is a frequently encountered peroxidaseproduced by white rot fungi (Urzúa et al., 1995, FEBS Letters 371:132-136). The enzyme catalyzes the oxidation of Mn²⁺ to Mn³⁺, which inturn can oxidize residues present in lignin. The peroxidase has acatalytic cycle involving a 2-electron oxidation of its heme by hydrogenperoxide to form compound I. Compound I can be reduced by Mn(II) or aphenolic substrate to compound II, the one electron form of the enzyme.The best reducing substrate for compounds I and II is Mn(II), a metalnaturally present in wood. The Mn(III) formed oxidizes other substrates.

Organic acids such as oxalate, glyoxylate and lactate are known to havean important role in the mechanism of manganese-dependent peroxidase andlignin degradation. Mn(III) is stripped from the enzyme by organicacids, and the produced Mn(III)-organic acid complex acts as adiffusible mediator in the oxidation of lignin by manganese-dependentperoxidase. Mn(III) can also oxidize organic acids, yielding radicals.The organic acids may also be supplied from the degradation of ligninand by microorganisms.

Several pathways leading to oxalate have been elucidated. One involvesthe cleavage of oxaloacetate by oxaloacetate hydrolase to form oxalicacid and acetate (Kubicek et al., 1988, Appl. Environ. Microb. 55:633-637). Another pathway involves the oxidation of glyoxylate to oxalicacid by the enzyme NAD-glyoxylate dehydrogenase (Balmforth and Thomson,1984, Biochem. J. 218: 341-342).

WO 2000/50576 describes an Aspergillus niger oxaloacetate hydrolase geneand fungal host cells made deficient in such a gene.

There is a need in the art for improving the lignin degradative abilityof strains by increasing the production of enzymes involved in degradinglignin. For example, Ceriporiopsis subvermispora is currently used forbiopulping and an engineered strain thereof with an improved lignindegradation capability would be particularly advantageous. Pretreatmentof wood chips with Ceriporiopsis subvermispora prior to mechanicalpulping has been shown to reduce energy consumption by 30-45% (Messnerand Srebotnik 1994, FEMS Microbiology Rev. 13: 351-364; Scott andSwaney, 1998, TAPPI J. 81: 153-175; Scott et al, 2000, RecentDevelopments in Biopulping Technology at Madison, Wis. In: L Viikari andR Lantto, ed. Progress in Biotechnology 21: Biotechnology in the Pulpand Paper Industry: 8^(th) ICBPPI Meeting. Amsterdam: Elsevier ScienceB.V. pp 61-71.). While the mechanism(s) of biopulping are unknown,Ceriporiopsis subvermispora colonization of wood appears to involve atleast two stages: an initial rapid colonization which softens woodwithout affecting fiber strength or lignin content, and later anefficient depolymerization and mineralization of lignin (Blanchette etal., 1988, Biomass 15: 93-101; Blanchette, 1991, Annu. Rev. Phytopathol.29: 381-398; Blanchette, 1994, Can. J. Botany 73: S999-S1010; Blanchetteet al., 1997, J. Biotechnology 53: 203-213; Breen and Singleton, 1999,Curr. Opinion Biotech. 10: 252-258).

It is an object of the present invention to provide improvedpolypeptides having oxaloacetate hydrolase activity and nucleic acidsencoding the polypeptides.

SUMMARY OF THE INVENTION

The present invention relates to isolated polypeptides havingoxaloacetate hydrolase activity selected from the group consisting of:

-   -   (a) a polypeptide having an amino acid sequence which has at        least 70% identity with SEQ ID NO: 2;    -   (b) a polypeptide which is encoded by a nucleotide sequence        which hybridizes under at least medium stringency conditions        with (i) SEQ ID NO: 1, (ii) the cDNA sequence contained in SEQ        ID NO: 1, or (iii) a complementary strand of (i) or (ii); and    -   (c) a variant comprising a conservative substitution, deletion,        and/or insertion of one or more amino acids of SEQ ID NO: 2.

The present invention also relates to isolated polynucleotides encodingpolypeptides having oxaloacetate hydrolase activity, selected from thegroup consisting of:

-   -   (a) a polynucleotide encoding a polypeptide having an amino acid        sequence which has at least 70% identity with SEQ ID NO: 2;    -   (b) a polynucleotide having at least 70% identity with SEQ ID        NO: 1; and    -   (c) a polynucleotide which hybridizes under at least medium        stringency conditions with (i) SEQ ID NO: 1, (ii) the cDNA        sequence contained in SEQ ID NO: 1, or (iii) a complementary        strand of (i) or (ii).

The present invention also relates to nucleic acid constructs,recombinant expression vectors, and recombinant host cells comprisingthe polynucleotides.

The present invention also relates to methods for producing suchpolypeptides having oxaloacetate hydrolase activity comprising (a)cultivating a recombinant host cell comprising a nucleic acid constructcomprising a polynucleotide encoding the polypeptide under conditionsconducive for production of the polypeptide; and (b) recovering thepolypeptide.

The present invention further relates to methods of delignifying amaterial comprising (a) treating the material with a microorganismcomprising a nucleic acid construct of the invention, and (b) recoveringthe treated material.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the genomic DNA sequence and the deduced amino acidsequence of a Ceriporiopsis subvermispora FPL 104807SS-5 (ForestProducts Laboratory, Madison, Wis.) oxaloacetate hydrolase (SEQ ID NOs:1 and 2, respectively).

FIG. 2 shows a restriction map of pBM115a.

Definitions

Oxaloacetate hydrolase activity: The term “oxaloacetate hydrolaseactivity” is defined herein as an oxaloacetate acetylhydrolase activitywhich catalyzes the hydrolysis of oxaloacetate to oxalic acid andacetate. Oxaloacetate hydrolases are classified as belonging to EC3.7.1.1. For purposes of the present invention, oxaloacetate hydrolaseactivity is determined according to the procedure described by Lenz etal., 1976, Eur. J. Biochem. 65: 225-236. One unit of oxaloacetatehydrolase activity is defined as 1.0 μmole of oxalic acid produced perminute at 30° C., pH 7.5.

The polypeptides of the present invention have at least 20%, preferablyat least 40%, more preferably at least 50%, more preferably at least60%, more preferably at least 70%, more preferably at least 80%, evenmore preferably at least 90%, most preferably at least 95%, and evenmost preferably at least 100% of the oxaloacetate hydrolase activity ofthe polypeptide consisting of the amino acid sequence shown as SEQ IDNO: 2.

Isolated polypeptide: The term “isolated polypeptide” as used hereinrefers to a polypeptide which is at least 20% pure, preferably at least40% pure, more preferably at least 60% pure, even more preferably atleast 80% pure, most preferably at least 90% pure, and even mostpreferably at least 95% pure, as determined by SDS-PAGE.

Substantially pure polypeptide: The term “substantially purepolypeptide” denotes herein a polypeptide preparation which contains atmost 10%, preferably at most 8%, more preferably at most 6%, morepreferably at most 5%, more preferably at most 4%, at most 3%, even morepreferably at most 2%, most preferably at most 1%, and even mostpreferably at most 0.5% by weight of other polypeptide material withwhich it is natively associated. It is, therefore, preferred that thesubstantially pure polypeptide is at least 92% pure, preferably at least94% pure, more preferably at least 95% pure, more preferably at least96% pure, more preferably at least 96% pure, more preferably at least97% pure, more preferably at least 98% pure, even more preferably atleast 99%, most preferably at least 99.5% pure, and even most preferably100% pure by weight of the total polypeptide material present in thepreparation.

The polypeptides of the present invention are preferably in asubstantially pure form. In particular, it is preferred that thepolypeptides are in “essentially pure form”, i.e., that the polypeptidepreparation is essentially free of other polypeptide material with whichit is natively associated. This can be accomplished, for example, bypreparing the polypeptide by means of well-known recombinant methods orby classical purification methods.

Herein, the term “substantially pure polypeptide” is synonymous with theterms “isolated polypeptide” and “polypeptide in isolated form.”

Identity: The relatedness between two amino acid sequences or betweentwo nucleotide sequences is described by the parameter “identity”.

For purposes of the present invention, the degree of identity betweentwo amino acid sequences is determined by the Clustal method (Higgins,1989, CABIOS 5: 151-153) using the LASERGENE™ MEGALIGN™ software(DNASTAR, Inc., Madison, Wis.) with an identity table and the followingmultiple alignment parameters: Gap penalty of 10 and gap length penaltyof 10. Pairwise alignment parameters are Ktuple=1, gap penalty=3,windows=5, and diagonals=5.

For purposes of the present invention, the degree of identity betweentwo nucleotide sequences is determined by the Wilbur-Lipman method(Wilbur and Lipman, 1983, Proceedings of the National Academy of ScienceUSA 80: 726-730) using the LASERGENE™ MEGALIGN™ software (DNASTAR, Inc.,Madison, Wis.) with an identity table and the following multiplealignment parameters: Gap penalty of 10 and gap length penalty of 10.Pairwise alignment parameters are Ktuple=3, gap penalty=3, andwindows=20.

Polypeptide Fragment: The term “polypeptide fragment” is defined hereinas a polypeptide having one or more amino acids deleted from the aminoand/or carboxyl terminus of SEQ ID NO: 2 or a homologous sequencethereof, wherein the fragment has oxaloacetate hydrolase activity.Preferably, a fragment contains at least 320 amino acid residues, morepreferably at least 335 amino acid residues, and most preferably atleast 350 amino acid residues.

Subsequence: The term “subsequence” is defined herein as a nucleotidesequence having one or more nucleotides deleted from the 5′ and/or 3′end of SEQ ID NO: 1 or a homologous sequence thereof, wherein thesubsequence encodes a polypeptide fragment having oxaloacetate hydrolaseactivity. Preferably, a subsequence contains at least 960 nucleotides,more preferably at least 1005 nucleotides, and most preferably at least1050 nucleotides.

Allelic variant: The term “allelic variant” denotes herein any of two ormore alternative forms of a gene occupying the same chromosomal locus.Allelic variation arises naturally through mutation, and may result inpolymorphism within populations. Gene mutations can be silent (no changein the encoded polypeptide) or may encode polypeptides having alteredamino acid sequences. An allelic variant of a polypeptide is apolypeptide encoded by an allelic variant of a gene.

Isolated polynucleotide: The term “isolated polynucleotide” as usedherein refers to a polynucleotide which is at least 20% pure, preferablyat least 40% pure, more preferably at least 60% pure, even morepreferably at least 80% pure, most preferably at least 90% pure, andeven most preferably at least 95% pure, as determined by agaroseelectrophoresis.

Substantially pure polynucleotide: The term “substantially purepolynucleotide” as used herein refers to a polynucleotide preparationfree of other extraneous or unwanted nucleotides and in a form suitablefor use within genetically engineered protein production systems. Thus,a substantially pure polynucleotide contains at most 10%, preferably atmost 8%, more preferably at most 6%, more preferably at most 5%, morepreferably at most 4%, more preferably at most 3%, even more preferablyat most 2%, most preferably at most 1%, and even most preferably at most0.5% by weight of other polynucleotide material with which it isnatively associated. A substantially pure polynucleotide may, however,include naturally occurring 5′ and 3′ untranslated regions, such aspromoters and terminators. It is preferred that the substantially purepolynucleotide is at least 90% pure, preferably at least 92% pure, morepreferably at least 94% pure, more preferably at least 95% pure, morepreferably at least 96% pure, more preferably at least 97% pure, evenmore preferably at least 98% pure, most preferably at least 99%, andeven most preferably at least 99.5% pure by weight. The polynucleotidesof the present invention are preferably in a substantially pure form. Inparticular, it is preferred that the polynucleotides disclosed hereinare in “essentially pure form”, i.e., that the polynucleotidepreparation is essentially free of other polynucleotide material withwhich it is natively associated. Herein, the term “substantially purepolynucleotide” is synonymous with the terms “isolated polynucleotide”and “polynucleotide in isolated form.” The polynucleotides may be ofgenomic, cDNA, RNA, semisynthetic, synthetic origin, or any combinationsthereof.

cDNA: The term “cDNA” is defined herein as a DNA molecule which can beprepared by reverse transcription from a mature, spliced, mRNA moleculeobtained from a eukaryotic cell. cDNA lacks intron sequences that areusually present in the corresponding genomic DNA. The initial, primaryRNA transcript is a precursor to mRNA which is processed through aseries of steps before appearing as mature spliced mRNA. These stepsinclude the removal of intron sequences by a process called splicing.cDNA derived from mRNA lacks, therefore, any intron sequences.

Nucleic acid construct: The term “nucleic acid construct” as used hereinrefers to a nucleic acid molecule, either single- or double-stranded,which is isolated from a naturally occurring gene or which is modifiedto contain segments of nucleic acids in a manner that would nototherwise exist in nature. The term nucleic acid construct is synonymouswith the term “expression cassette” when the nucleic acid constructcontains the control sequences required for expression of a codingsequence of the present invention.

Control sequence: The term “control sequences” is defined herein toinclude all components, which are necessary or advantageous for theexpression of a polynucleotide encoding a polypeptide of the presentinvention. Each control sequence may be native or foreign to thenucleotide sequence encoding the polypeptide. Such control sequencesinclude, but are not limited to, a leader, polyadenylation sequence,propeptide sequence, promoter, signal peptide sequence, andtranscription terminator. At a minimum, the control sequences include apromoter, and transcriptional and translational stop signals. Thecontrol sequences may be provided with linkers for the purpose ofintroducing specific restriction sites facilitating ligation of thecontrol sequences with the coding region of the nucleotide sequenceencoding a polypeptide.

Operably linked: The term “operably linked” denotes herein aconfiguration in which a control sequence is placed at an appropriateposition relative to the coding sequence of the polynucleotide sequencesuch that the control sequence directs the expression of the codingsequence of a polypeptide.

Coding sequence: When used herein the term “coding sequence” means anucleotide sequence, which directly specifies the amino acid sequence ofits protein product. The boundaries of the coding sequence are generallydetermined by an open reading frame, which usually begins with the ATGstart codon or alternative start codons such as GTG and TTG. The codingsequence may a DNA, cDNA, or recombinant nucleotide sequence.

Expression: The term “expression” includes any step involved in theproduction of the polypeptide including, but not limited to,transcription, post-transcriptional modification, translation,post-translational modification, and secretion.

Expression vector: The term “expression vector” is defined herein as alinear or circular DNA molecule that comprises a polynucleotide encodinga polypeptide of the invention, and which is operably linked toadditional nucleotides that provide for its expression.

Host cell: The term “host cell”, as used herein, includes any cell typewhich is susceptible to transformation, transfection, transduction, andthe like with a nucleic acid construct or expression vector comprising apolynucleotide of the present invention.

Modification: The term “modification” means herein any chemicalmodification of the polypeptide consisting of the SEQ ID NO: 2 or ahomologous sequence thereof as well as genetic manipulation of the DNAencoding that polypeptide. The modification can be substitution,deletion and/or insertion of one or more amino acids as well asreplacements of one or more amino acid side chains.

Artificial variant: When used herein, the term “artificial variant”means a polypeptide having oxaloacetate hydrolase activity produced byan organism expressing a modified nucleotide sequence of SEQ ID NO: 1 ora homologous sequence thereof. The modified nucleotide sequence isobtained through human intervention by modification of the nucleotidesequence disclosed in SEQ ID NO: 1 or a homologous sequence thereof.

DETAILED DESCRIPTION OF THE INVENTION

Polypeptides Having Oxaloacetate Hydrolase Activity

In a first aspect, the present invention relates to isolatedpolypeptides having an amino acid sequence which has a degree ofidentity to SEQ ID NO: 2 of at least 70%, preferably at least 75%, morepreferably at least 80%, more preferably at least 85%, even morepreferably at least 90%, most preferably at least 95%, and even mostpreferably at least 97%, which have oxaloacetate hydrolase activity(hereinafter “homologous polypeptides”). In a preferred aspect, thehomologous polypeptides have an amino acid sequence which differs by tenamino acids, preferably by five amino acids, more preferably by fouramino acids, even more preferably by three amino acids, most preferablyby two amino acids, and even most preferably by one amino acid from SEQID NO: 2.

A polypeptide of the present invention preferably comprises the aminoacid sequence of SEQ ID NO: 2 or an allelic variant thereof; or afragment thereof that has oxaloacetate hydrolase activity. In apreferred aspect, a polypeptide comprises the amino acid sequence of SEQID NO: 2. In another preferred aspect, a polypeptide consists of theamino acid sequence of SEQ ID NO: 2 or an allelic variant thereof; or afragment thereof that has oxaloacetate hydrolase activity. In anotherpreferred aspect, a polypeptide consists of the amino acid sequence ofSEQ ID NO: 2.

In a second aspect, the present invention relates to isolatedpolypeptides having oxaloacetate hydrolase activity which are encoded bypolynucleotides which hybridize under very low stringency conditions,preferably low stringency conditions, more preferably medium stringencyconditions, more preferably medium-high stringency conditions, even morepreferably high stringency conditions, and most preferably very highstringency conditions with (i) SEQ ID NO: 1, (ii) the cDNA sequencecontained in SEQ ID NO: 1, (iii) a subsequence of (i) or (ii), or (iv) acomplementary strand of (i), (ii), or (iii) (J. Sambrook, E. F. Fritsch,and T. Maniatus, 1989, Molecular Cloning, A Laboratory Manual, 2dedition, Cold Spring Harbor, N.Y.). A subsequence of SEQ ID NO: 1contains at least 100 contiguous nucleotides or preferably at least 200continguous nucleotides. Moreover, the subsequence may encode apolypeptide fragment which has oxaloacetate hydrolase activity.

The nucleotide sequence of SEQ ID NO: 1 or a subsequence thereof, aswell as the amino acid sequence of SEQ ID NO: 2 or a fragment thereof,may be used to design a nucleic acid probe to identify and clone DNAencoding polypeptides having oxaloacetate hydrolase activity fromstrains of different genera or species according to methods well knownin the art. In particular, such probes can be used for hybridizationwith the genomic or CDNA of the genus or species of interest, followingstandard Southern blotting procedures, in order to identify and isolatethe corresponding gene therein. Such probes can be considerably shorterthan the entire sequence, but should be at least 14, preferably at least25, more preferably at least 35, and most preferably at least 70nucleotides in length. It is, however, preferred that the nucleic acidprobe is at least 100 nucleotides in length. For example, the nucleicacid probe may be at least 200 nucleotides, preferably at least 300nucleotides, more preferably at least 400 nucleotides, or mostpreferably at least 500 nucleotides in length. Even longer probes may beused, e.g., nucleic acid probes which are at least 600 nucleotides, atleast preferably at least 700 nucleotides, more preferably at least 800nucleotides, or most preferably at least 900 nucleotides in length. BothDNA and RNA probes can be used. The probes are typically labeled fordetecting the corresponding gene (for example, with ³²p, ³H, ³⁵S,biotin, or avidin). Such probes are encompassed by the presentinvention.

A genomic DNA or cDNA library prepared from such other organisms may,therefore, be screened for DNA which hybridizes with the probesdescribed above and which encodes a polypeptide having oxaloacetatehydrolase activity. Genomic or other DNA from such other organisms maybe separated by agarose or polyacrylamide gel electrophoresis, or otherseparation techniques. DNA from the libraries or the separated DNA maybe transferred to and immobilized on nitrocellulose or other suitablecarrier material. In order to identify a clone or DNA which ishomologous with SEQ ID NO: 1 or a subsequence thereof, the carriermaterial is used in a Southern blot.

For purposes of the present invention, hybridization indicates that thenucleotide sequence hybridizes to a labeled nucleic acid probecorresponding to the nucleotide sequence shown in SEQ ID NO: 1, the cDNAsequence contained in SEQ ID NO: 1, its complementary strand, or asubsequence thereof, under very low to very high stringency conditions.Molecules to which the nucleic acid probe hybridizes under theseconditions can be detected using X-ray film.

In a preferred aspect, the nucleic acid probe is SEQ ID NO: 1 or thecomplementary strand thereof. In another preferred aspect, the nucleicacid probe is a polynucleotide sequence which encodes the polypeptide ofSEQ ID NO: 2, or a subsequence thereof. In another preferred aspect, thenucleic acid probe is the polynucleotide sequence contained in plasmidpBM115a which is contained in Escherichia coli NRRL B-30669, wherein thepolynucleotide sequence thereof encodes a polypeptide havingoxaloacetate hydrolase activity.

For long probes of at least 100 nucleotides in length, very low to veryhigh stringency conditions are defined as prehybridization andhybridization at 42° C. in 5×SSPE, 0.3% SDS, 200 μg/ml sheared anddenatured salmon sperm DNA, and either 25% formamide for very low andlow stringencies, 35% formamide for medium and medium-high stringencies,or 50% formamide for high and very high stringencies, following standardSouthern blotting procedures for 12 to 24 hours optimally.

For long probes of at least 100 nucleotides in length, the carriermaterial is finally washed three times each for 15 minutes using 2×SSC,0.2% SDS preferably at least at 45° C. (very low stringency), morepreferably at least at 50° C. (low stringency), more preferably at leastat 55° C. (medium stringency), more preferably at least at 60° C.(medium-high stringency), even more preferably at least at 65° C. (highstringency), and most preferably at least at 70° C. (very highstringency).

For short probes which are about 15 nucleotides to about 70 nucleotidesin length, stringency conditions are defined as prehybridization,hybridization, and washing post-hybridization at about 5° C. to about10° C. below the calculated T_(m) using the calculation according toBolton and McCarthy (1962, Proceedings of the National Academy ofSciences USA 48:1390) in 0.9 M NaCl, 0.09 M Tris-HCl pH 7.6, 6 mM EDTA,0.5% NP-40, 1×Denhardt's solution, 1 mM sodium pyrophosphate, 1 mMsodium monobasic phosphate, 0.1 mM ATP, and 0.2 mg of yeast RNA per mlfollowing standard Southern blotting procedures for 12 to 24 hoursoptimally.

For short probes which are about 15 nucleotides to about 70 nucleotidesin length, the carrier material is washed once in 6×SCC plus 0.1% SDSfor 15 minutes and twice each for 15 minutes using 6×SSC at 5° C. to 10°C. below the calculated T_(m).

Under salt-containing hybridization conditions, the effective T_(m) iswhat controls the degree of identity required between the probe and thefilter bound DNA for successful hybridization. The effective T_(m) maybe determined using the formula below to determine the degree ofidentity required for two DNAs to hybridize under various stringencyconditions.Effective T_(m)=81.5+16.6(log M[Na⁺])+0.41(% G+C)−0.72(% formamide)

The G+C content of SEQ ID NO: 1 is 58.3%. For medium stringency, theformamide is 35% and the Na⁺ concentration for 5×SSPE is 0.75 M.Applying this formula to these values, the Effective T_(m) is 78.13° C.

Another relevant relationship is that a 1% mismatch of two DNAs lowersthe T_(m) by 1.4° C. To determine the degree of identity required fortwo DNAs to hybridize under medium stringency conditions at 42° C., thefollowing formula is used:% Homology=100−[(Effective T_(m)−Hybridization Temperature)/1.4]

Applying this formula to the values, the degree of identity required fortwo DNAs to hybridize under medium stringency conditions at 42° C. is74.19%.

In a third aspect, the present invention relates to artificial variantscomprising a conservative substitution, deletion, and/or insertion ofone or more amino acids of SEQ ID NO: 2 or a homologous sequencethereof. Preferably, amino acid changes are of a minor nature, that isconservative amino acid substitutions or insertions that do notsignificantly affect the folding and/or activity of the protein; smalldeletions, typically of one to about 30 amino acids; small amino- orcarboxyl-terminal extensions, such as an amino-terminal methionineresidue; a small linker peptide of up to about 20-25 residues; or asmall extension that facilitates purification by changing net charge oranother function, such as a poly-histidine tract, an antigenic epitopeor a binding domain.

Examples of conservative substitutions are within the group of basicamino acids (arginine, lysine and histidine), acidic amino acids(glutamic acid and aspartic acid), polar amino acids (glutamine andasparagine), hydrophobic amino acids (leucine, isoleucine and valine),aromatic amino acids (phenylalanine, tryptophan and tyrosine), and smallamino acids (glycine, alanine, serine, threonine and methionine). Aminoacid substitutions which do not generally alter specific activity areknown in the art and are described, for example, by H. Neurath and R. L.Hill, 1979, In, The Proteins, Academic Press, New York. The mostcommonly occurring exchanges are Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser,Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Tyr/Phe, Ala/Pro, Lys/Arg,Asp/Asn, Leu/Ile, Leu/Val, Ala/Glu, and Asp/Gly.

In addition to the 20 standard amino acids, non-standard amino acids(such as 4-hydroxyproline, 6-N-methyl lysine, 2-aminoisobutyric acid,isovaline, and alpha-methyl serine) may be substituted for amino acidresidues of a wild-type polypeptide. A limited number ofnon-conservative amino acids, amino acids that are not encoded by thegenetic code, and unnatural amino acids may be substituted for aminoacid residues. “Unnatural amino acids” have been modified after proteinsynthesis, and/or have a chemical structure in their side chain(s)different from that of the standard amino acids. Unnatural amino acidscan be chemically synthesized, and preferably, are commerciallyavailable, and include pipecolic acid, thiazolidine carboxylic acid,dehydroproline, 3- and 4-methylproline, and 3,3-dimethylproline.

Alternatively, the amino acid changes are of such a nature that thephysico-chemical properties of the polypeptides are altered. Forexample, amino acid changes may improve the thermal stability of thepolypeptide, alter the substrate specificity, change the pH optimum, andthe like.

Essential amino acids in the parent polypeptide can be identifiedaccording to procedures known in the art, such as site-directedmutagenesis or alanine-scanning mutagenesis (Cunningham and Wells, 1989,Science 244: 1081-1085). In the latter technique, single alaninemutations are introduced at every residue in the molecule, and theresultant mutant molecules are tested for biological activity (i.e.,oxaloacetate hydrolase activity) to identify amino acid residues thatare critical to the activity of the molecule. See also, Hilton et al.,1996, J. Biol. Chem. 271: 4699-4708. The active site of the enzyme orother biological interaction can also be determined by physical analysisof structure, as determined by such techniques as nuclear magneticresonance, crystallography, electron diffraction, or photoaffinitylabeling, in conjunction with mutation of putative contact site aminoacids. See, for example, de Vos et al., 1992, Science 255: 306-312;Smith et al., 1992, J. Mol. Biol. 224: 899-904; Wlodaver et al., 1992,FEBS Lett. 309:59-64. The identities of essential amino acids can alsobe inferred from analysis of identities with polypeptides which arerelated to a polypeptide according to the invention.

Single or multiple amino acid substitutions can be made and tested usingknown methods of mutagenesis, recombination, and/or shuffling, followedby a relevant screening procedure, such as those disclosed byReidhaar-Olson and Sauer, 1988, Science 241: 53-57; Bowie and Sauer,1989, Proc. Natl. Acad. Sci. USA 86: 2152-2156; WO 95/17413; or WO95/22625. Other methods that can be used include error-prone PCR, phagedisplay (e.g., Lowman et al., 1991, Biochem. 30:10832-10837; U.S. Pat.No. 5,223,409; WO 92/06204), and region-directed mutagenesis (Derbyshireet al., 1986, Gene 46:145; Ner et al., 1988, DNA 7:127).

Mutagenesis/shuffling methods can be combined with high-throughput,automated screening methods to detect activity of cloned, mutagenizedpolypeptides expressed by host cells. Mutagenized DNA molecules thatencode active polypeptides can be recovered from the host cells andrapidly sequenced using standard methods in the art. These methods allowthe rapid determination of the importance of individual amino acidresidues in a polypeptide of interest, and can be applied topolypeptides of unknown structure.

The total number of amino acid substitutions, deletions and/orinsertions of SEQ ID NO: 2 is 10, preferably 9, more preferably 8, morepreferably 7, more preferably 6, more preferably 5, more preferably 4,even more preferably 3, most preferably 2, and even most preferably 1.

Sources of Polypeptides Having Oxaloacetate Hydrolase Activity

A polypeptide of the present invention may be obtained frommicroorganisms of any genus. For purposes of the present invention, theterm “obtained from” as used herein in connection with a given sourceshall mean that the polypeptide encoded by a nucleotide sequence isproduced by the source or by a strain in which the nucleotide sequencefrom the source has been inserted. In a preferred aspect, thepolypeptide obtained from a given source is secreted extracellularly.

A polypeptide of the present invention may be a fungal polypeptide, andmore preferably a yeast polypeptide such as a Candida, Kluyveromyces,Pichia, Saccharomyces, Schizosaccharomyces, or Yarrowia polypeptide; ormore preferably a filamentous fungal polypeptide such as an Acremonium,Aspergillus, Aureobasidium, Cryptococcus, Filibasidium, Fusarium,Humicola, Magnaporthe, Mucor, Myceliophthora, Neocallimastix,Neurospora, Paecilomyces, Penicillium, Piromyces, Schizophyllum,Talaromyces, Thermoascus, Thielavia, Tolypocladium, or Trichodermapolypeptide.

In a preferred aspect, the polypeptide is a Saccharomycescarlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus,Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomycesnorbensis, or Saccharomyces oviformis polypeptide having oxaloacetatehydrolase activity.

In another preferred aspect, the polypeptide is an Aspergillusaculeatus, Aspergillus awamori, Aspergillus fumigatus, Aspergillusfoetidus, Aspergillus japonicus, Aspergillus nidulans, Aspergillusniger, Aspergillus oryzae, Fusarium bactridioides, Fusarium cerealis,Fusarium crookwellense, Fusarium culmorum, Fusarium graminearum,Fusarium graminum, Fusarium heterosporum, Fusarium negundi, Fusariumoxysporum, Fusarium reticulatum, Fusarium roseum, Fusarium sambucinum,Fusarium sarcochroum, Fusarium sporotrichioides, Fusarium sulphureum,Fusarium torulosum, Fusarium trichothecioides, Fusarium venenatum,Humicola insolens, Humicola lanuginosa, Mucor miehei, Myceliophthorathermophila, Neurospora crassa, Penicillium purpurogenum, Trichodermaharzianum, Trichoderma koningii, Trichoderma longibrachiatum,Trichoderma reesei, or Trichoderma viride polypeptide.

In another preferred aspect, the polypeptide is a Ceriporiopsisaneirina, Ceriporiopsis aneirina, Ceriporiopsis caregiea, Ceriporiopsisgilvescens, Ceriporiopsis pannocinta, Ceriporlopsis rivulosa,Ceriporiopsis subrufa, or Cerlporiopsis subvermispora polypeptide.

In a more preferred aspect, the polypeptide is a Ceriporiopsissubvermispora polypeptide, and most preferably a Ceriporiopsissubvermispora FPL 104807SS-5 (Forest Products Laboratory, Madison, Wis.)polypeptide, e.g., the polypeptide of SEQ ID NO: 2.

It will be understood that for the aforementioned species the inventionencompasses both the perfect and imperfect states, and other taxonomicequivalents, e.g., anamorphs, regardless of the species name by whichthey are known. Those skilled in the art will readily recognize theidentity of appropriate equivalents.

Strains of these species are readily accessible to the public in anumber of culture collections, such as the American Type CultureCollection (ATCC), Deutsche Sammlung von Mikroorganismen undZellkulturen GmbH (DSM), Centraalbureau Voor Schimmelcultures (CBS), andAgricultural Research Service Patent Culture Collection, NorthernRegional Research Center (NRRL).

Furthermore, such polypeptides may be identified and obtained from othersources including microorganisms isolated from nature (e.g., soil,composts, water, etc.) using the above-mentioned probes. Techniques forisolating microorganisms from natural habitats are well known in theart. The polynucleotide may then be obtained by similarly screening agenomic or cDNA library of such a microorganism. Once a polynucleotidesequence encoding a polypeptide has been detected with the probe(s), thepolynucleotide can be isolated or cloned by utilizing techniques whichare well known to those of ordinary skill in the art (see, e.g.,Sambrook et al., 1989, supra).

Polypeptides of the present invention also include fused polypeptides orcleavable fusion polypeptides in which another polypeptide is fused atthe N-terminus or the C-terminus of the polypeptide or fragment thereof.A fused polypeptide is produced by fusing a nucleotide sequence (or aportion thereof) encoding another polypeptide to a nucleotide sequence(or a portion thereof) of the present invention. Techniques forproducing fusion polypeptides are known in the art, and include ligatingthe coding sequences encoding the polypeptides so that they are in frameand that expression of the fused polypeptide is under control of thesame promoter(s) and terminator.

Polynucleotides

The present invention also relates to isolated polynucleotides having anucleotide sequence which encode a polypeptide of the present invention.In a preferred aspect, the nucleotide sequence is set forth in SEQ IDNO: 1. In another more preferred aspect, the nucleotide sequence is thesequence contained in plasmid pBM1l5a that is contained in Escherichiacoli NRRL B-30669. The present invention also encompasses nucleotidesequences which encode a polypeptide having the amino acid sequence ofSEQ ID NO: 2, which differ from SEQ ID NO: 1 by virtue of the degeneracyof the genetic code. The present invention also relates to subsequencesof SEQ ID NO: 1 which encode fragments of SEQ ID NO: 2 that haveoxaloacetate hydrolase activity.

The present invention also relates to mutant polynucleotides comprisingat least one mutation in the polypeptide coding sequence of SEQ ID NO:1, in which the mutant nucleotide sequence encodes a polypeptide whichconsists of SEQ ID NO: 2.

The techniques used to isolate or clone a polynucleotide encoding apolypeptide are known in the art and include isolation from genomic DNA,preparation from cDNA, or a combination thereof. The cloning of thepolynucleotides of the present invention from such genomic DNA can beeffected, e.g., by using the well known polymerase chain reaction (PCR)or antibody screening of expression libraries to detect cloned DNAfragments with shared structural features. See, e.g., Innis et al.,1990, PCR: A Guide to Methods and Application, Academic Press, New York.Other nucleic acid amplification procedures such as ligase chainreaction (LCR), ligated activated transcription (LAT) and nucleotidesequence-based amplification (NASBA) may be used. The polynucleotidesmay be cloned from a strain of Ceriporiopsis, or another or relatedorganism and thus, for example, may be an allelic or species variant ofthe polypeptide encoding region of the nucleotide sequence.

The present invention also relates to polynucleotides having nucleotidesequences which have a degree of identity to SEQ ID NO: 1 of at least70%, preferably at least 75%, more preferably at least 80%, morepreferably at least 85%, more preferably at least 90%, even morepreferably at least 95%, and most preferably at least 97% identity,which encode an active polypeptide.

Modification of a nucleotide sequence encoding a polypeptide of thepresent invention may be necessary for the synthesis of polypeptidessubstantially similar to the polypeptide. The term “substantiallysimilar” to the polypeptide refers to non-naturally occurring forms ofthe polypeptide. These polypeptides may differ in some engineered wayfrom the polypeptide isolated from its native source, e.g., artificialvariants that differ in specific activity, thermostability, pH optimum,or the like. The variant sequence may be constructed on the basis of thenucleotide sequence presented as the polypeptide encoding region of SEQID NO: 1, e.g., a subsequence thereof, and/or by introduction ofnucleotide substitutions which do not give rise to another amino acidsequence of the polypeptide encoded by the nucleotide sequence, butwhich correspond to the codon usage of the host organism intended forproduction of the enzyme, or by introduction of nucleotide substitutionswhich may give rise to a different amino acid sequence. For a generaldescription of nucleotide substitution, see, e.g., Ford et al., 1991,Protein Expression and Purification 2: 95-107.

It will be apparent to those skilled in the art that such substitutionscan be made outside the regions critical to the function of the moleculeand still result in an active polypeptide. Amino acid residues essentialto the activity of the polypeptide encoded by an isolated polynucleotideof the invention, and therefore preferably not subject to substitution,may be identified according to procedures known in the art, such assite-directed mutagenesis or alanine-scanning mutagenesis (see, e.g.,Cunningham and Wells, 1989, Science 244: 1081-1085). In the lattertechnique, mutations are introduced at every positively charged residuein the molecule, and the resultant mutant molecules are tested foroxaloacetate hydrolase activity to identify amino acid residues that arecritical to the activity of the molecule. Sites of substrate-enzymeinteraction can also be determined by analysis of the three-dimensionalstructure as determined by such techniques as nuclear magnetic resonanceanalysis, crystallography or photoaffinity labelling (see, e.g., de Voset al., 1992, Science 255: 306-312; Smith et al., 1992, Journal ofMolecular Biology224: 899-904; Wlodaver et al., 1992, FEBS Letters 309:59-64).

The present invention also relates to isolated polynucleotides encodinga polypeptide of the present invention, which hybridize under very lowstringency conditions, preferably low stringency conditions, morepreferably medium stringency conditions, more preferably medium-highstringency conditions, even more preferably high stringency conditions,and most preferably very high stringency conditions with (i) SEQ ID NO:1, (ii) the cDNA sequence contained in SEQ ID NO: 1, or (iii) acomplementary strand of (i) or (ii); or allelic variants andsubsequences thereof (Sambrook et al., 1989, supra), as defined herein.

The present invention also relates to isolated polynucleotides obtainedby (a) hybridizing a population of DNA under very low, low, medium,medium-high, high, or very high stringency conditions with (i) SEQ IDNO: 1, (ii) the cDNA sequence contained in SEQ ID NO: 1, or (iii) acomplementary strand of (i) or (ii); and (b) isolating the hybridizingpolynucleotide, which encodes a polypeptide having oxaloacetatehydrolase activity.

Nucleic Acid Constructs

The present invention also relates to nucleic acid constructs comprisingan isolated polynucleotide of the present invention operably linked toone or more control sequences which direct the expression of the codingsequence in a suitable host cell under conditions compatible with thecontrol sequences.

An isolated polynucleotide encoding a polypeptide of the presentinvention may be manipulated in a variety of ways to provide forexpression of the polypeptide. Manipulation of the polynucleotide'ssequence prior to its insertion into a vector may be desirable ornecessary depending on the expression vector. The techniques formodifying polynucleotide sequences utilizing recombinant DNA methods arewell known in the art.

The control sequence may be an appropriate promoter sequence, anucleotide sequence which is recognized by a host cell for expression ofa polynucleotide encoding a polypeptide of the present invention. Thepromoter sequence contains transcriptional control sequences whichmediate the expression of the polypeptide. The promoter may be anynucleotide sequence which shows transcriptional activity in the hostcell of choice including mutant, truncated, and hybrid promoters, andmay be obtained from genes encoding extracellular or intracellularpolypeptides either homologous or heterologous to the host cell.

Examples of suitable promoters for directing the transcription of thenucleic acid constructs of the present invention in a filamentous fungalhost cell are promoters obtained from the genes for Aspergillus oryzaeTAKA amylase, Rhizomucor miehei aspartic proteinase, Aspergillus nigerneutral alpha-amylase, Aspergillus niger acid stable alpha-amylase,Aspergillus niger or Aspergillus awamori glucoamylase (glaA), Rhizomucormiehei lipase, Aspergillus oryzae alkaline protease, Aspergillus oryzaetriose phosphate isomerase, Aspergillus nidulans acetamidase, Fusariumvenenatum amylogrucosidase, Fusarium oxysporum trypsin-like protease (WO96/00787), Trichoderma reesei cellobiohydrolase I, Trichodermareeseicellobiohydrolase II, Trichoderma reeseiendoglucanase I,Trichoderma reesei endoglucanase II, Trichoderma reesei endoglucanaseIII, Trichoderma reesei endoglucanase IV, Trichoderma reeseiendoglucanase V, Trichoderma reesei xylanase I, Trichoderma reeseixylanase II, Trichoderma reesei beta-xylosidase, as well as the NA2-tpipromoter (a hybrid of the promoters from the genes for Aspergillus nigerneutral alpha-amylase and Aspergillus oryzae triose phosphateisomerase); and mutant, truncated, and hybrid promoters thereof.

In a yeast host, useful promoters are obtained from the genes forSaccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiaegalactokinase (GALl), Saccharomyces cerevisiae alcoholdehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH1,ADH2/GAP),Saccharomyces cerevisiae triose phosphate isomerase (TPI), Saccharomycescerevisiae metallothionine (CUP1), and Saccharomyces cerevisiae3-phosphoglycerate kinase. Other useful promoters for yeast host cellsare described by Romanos etal., 1992, Yeast8: 423-488.

The control sequence may also be a suitable transcription terminatorsequence, a sequence recognized by a host cell to terminatetranscription. The terminator sequence is operably linked to the 3′terminus of the nucleotide sequence encoding the polypeptide. Anyterminator which is functional in the host cell of choice may be used inthe present invention.

Preferred terminators for filamentous fungal host cells are obtainedfrom the genes for Aspergillus oryzae TAKA amylase, Aspergillus nigerglucoamylase, Aspergillus nidulans anthranilate synthase, Aspergillusniger alpha-glucosidase, and Fusarium oxysporum trypsin-like protease.

Preferred terminators for yeast host cells are obtained from the genesfor Saccharomyces cerevisiae enolase, Saccharomyces cerevisiaecytochrome C (CYC1), and Saccharomyces cerevisiaeglyceraldehyde-3-phosphate dehydrogenase. Other useful terminators foryeast host cells are described by Romanos et al., 1992, supra.

The control sequence may also be a suitable leader sequence, anontranslated region of an mRNA which is important for translation bythe host cell. The leader sequence is operably linked to the 5′ terminusof the nucleotide sequence encoding the polypeptide. Any leader sequencethat is functional in the host cell of choice may be used in the presentinvention.

Preferred leaders for filamentous fungal host cells are obtained fromthe genes for Aspergillus oryzae TAKA amylase and Aspergillus nidulanstriose phosphate isomerase.

Suitable leaders for yeast host cells are obtained from the genes forSaccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae3-phosphoglycerate kinase, Saccharomyces cerevisiae alpha-factor, andSaccharomyces cerevisiae alcoholdehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH2/GAP).

The control sequence may also be a polyadenylation sequence, a sequenceoperably linked to the 3′ terminus of the nucleotide sequence and which,when transcribed, is recognized by the host cell as a signal to addpolyadenosine residues to transcribed mRNA. Any polyadenylation sequencewhich is functional in the host cell of choice may be used in thepresent invention.

Preferred polyadenylation sequences for filamentous fungal host cellsare obtained from the genes for Aspergillus oryzae TAKA amylase,Aspergillus niger glucoamylase, Aspergillus nidulans anthranilatesynthase, Fusarium oxysporum trypsin-like protease, and Aspergillusniger alpha-glucosidase.

Useful polyadenylation sequences for yeast host cells are described byGuo and Sherman, 1995, Molecular Cellular Biology 15: 5983-5990.

The control sequence may also be a signal peptide coding region thatcodes for an amino acid sequence linked to the amino terminus of apolypeptide and directs the encoded polypeptide into the cell'ssecretory pathway. The 5′ end of the coding sequence of the nucleotidesequence may inherently contain a signal peptide coding region naturallylinked in translation reading frame with the segment of the codingregion which encodes the secreted polypeptide. Alternatively, the 5′ endof the coding sequence may contain a signal peptide coding region whichis foreign to the coding sequence. The foreign signal peptide codingregion may be required where the coding sequence does not naturallycontain a signal peptide coding region. Alternatively, the foreignsignal peptide coding region may simply replace the natural signalpeptide coding region in order to enhance secretion of the polypeptide.However, any signal peptide coding region which directs the expressedpolypeptide into the secretory pathway of a host cell of choice may beused in the present invention.

Effective signal peptide coding regions for filamentous fungal hostcells are the signal peptide coding regions obtained from the genes forAspergillus oryzae TAKA amylase, Aspergillus niger neutral amylase,Aspergillus niger glucoamylase, Rhizomucor miehei aspartic proteinase,Humicola insolens cellulase, Humicola insolens endoglucanase V, andHumicola lanuginosa lipase.

Useful signal peptides for yeast host cells are obtained from the genesfor Saccharomyces cerevisiae alpha-factor and Saccharomyces cerevisiaeinvertase. Other useful signal peptide coding regions are described byRomanos et al., 1992, supra.

The control sequence may also be a propeptide coding region that codesfor an amino acid sequence positioned at the amino terminus of apolypeptide. The resultant polypeptide is known as a proenzyme orpropolypeptide (or a zymogen in some cases). A propolypeptide isgenerally inactive and can be converted to a mature active polypeptideby catalytic or autocatalytic cleavage of the propeptide from thepropolypeptide. The propeptide coding region may be obtained from thegenes for Saccharomyces cerevisiae alpha-factor, Rhizomucor mieheiaspartic proteinase, and Myceliophthora thermophila laccase (WO95/33836).

Where both signal peptide and propeptide regions are present at theamino terminus of a polypeptide, the propeptide region is positionednext to the amino terminus of a polypeptide and the signal peptideregion is positioned next to the amino terminus of the propeptideregion.

It may also be desirable to add regulatory sequences which allow theregulation of the expression of the polypeptide relative to the growthof the host cell. Examples of regulatory systems are those which causethe expression of the gene to be turned on or off in response to achemical or physical stimulus, including the presence of a regulatorycompound. In yeast, the ADH2 system or GAL1 system may be used. Infilamentous fungi, the TAKA alpha-amylase promoter, Aspergillus nigerglucoamylase promoter, and Aspergillus olyzae glucoamylase promoter maybe used as regulatory sequences. Other examples of regulatory sequencesare those which allow for gene amplification. In eukaryotic systems,these include the dihydrofolate reductase gene which is amplified in thepresence of methotrexate, and the metallothionein genes which areamplified with heavy metals. In these cases, the nucleotide sequenceencoding the polypeptide would be operably linked with the regulatorysequence.

Expression Vectors

The present invention also relates to recombinant expression vectorscomprising a polynucleotide of the present invention, a promoter, andtranscriptional and translational stop signals. The various nucleicacids and control sequences described herein may be joined together toproduce a recombinant expression vector which may include one or moreconvenient restriction sites to allow for insertion or substitution ofthe nucleotide sequence encoding the polypeptide at such sites.Alternatively, a nucleotide sequence of the present invention may beexpressed by inserting the nucleotide sequence or a nucleic acidconstruct comprising the sequence into an appropriate vector forexpression. In creating the expression vector, the coding sequence islocated in the vector so that the coding sequence is operably linkedwith the appropriate control sequences for expression.

The recombinant expression vector may be any vector (e.g., a plasmid orvirus) which can be conveniently subjected to recombinant DNA proceduresand can bring about expression of the nucleotide sequence. The choice ofthe vector will typically depend on the compatibility of the vector withthe host cell into which the vector is to be introduced. The vectors maybe linear or closed circular plasmids.

The vector may be an autonomously replicating vector, i.e., a vectorwhich exists as an extrachromosomal entity, the replication of which isindependent of chromosomal replication, e.g., a plasmid, anextrachromosomal element, a minichromosome, or an artificial chromosome.The vector may contain any means for assuring self-replication.Alternatively, the vector may be one which, when introduced into thehost cell, is integrated into the genome and replicated together withthe chromosome(s) into which it has been integrated. Furthermore, asingle vector or plasmid or two or more vectors or plasmids whichtogether contain the total DNA to be introduced into the genome of thehost cell, or a transposon may be used.

The vectors of the present invention preferably contain one or moreselectable markers which permit easy selection of transformed,transfected, transduced, or the like cells. A selectable marker is agene the product of which provides for biocide or viral resistance,resistance to heavy metals, prototrophy to auxotrophs, and the like.

Suitable markers for yeast host cells are ADE2, HIS3, LEU2, LYS2, MET3,TRP1, and URA3. Selectable markers for use in a filamentous fungal hostcell include, but are not limited to, amdS (acetamidase), argB(ornithine carbamoyltransferase), bar (phosphinothricinacetyltransferase), hph (hygromycin phosphotransferase), niaD (nitratereductase), pyrG (orotidine-5′-phosphate decarboxylase), sC (sulfateadenyltransferase), and trpC (anthranilate synthase), as well asequivalents thereof. Preferred for use in an Aspergillus cell are theamdS and pyrG genes of Aspergillus nidulans or Aspergillus oryzae andthe bargene of Streptomyces hygroscopicus.

The vectors of the present invention preferably contain an element(s)that permits integration of the vector into the host cell's genome orautonomous replication of the vector in the cell independent of thegenome.

For integration into the host cell genome, the vector may rely on thepolynucleotide's sequence encoding the polypeptide or any other elementof the vector for integration into the genome by homologous ornonhomologous recombination. Alternatively, the vector may containadditional nucleotide sequences for directing integration by homologousrecombination into the genome of the host cell at a precise location(s)in the chromosome(s). To increase the likelihood of integration at aprecise location, the integrational elements should preferably contain asufficient number of nucleic acids, such as 100 to 10,000 base pairs,preferably 400 to 10,000 base pairs, and most preferably 800 to 10,000base pairs, which have a high degree of identity with the correspondingtarget sequence to enhance the probability of homologous recombination.The integrational elements may be any sequence that is homologous withthe target sequence in the genome of the host cell. Furthermore, theintegrational elements may be non-encoding or encoding nucleotidesequences. On the other hand, the vector may be integrated into thegenome of the host cell by non-homologous recombination.

For autonomous replication, the vector may further comprise an origin ofreplication enabling the vector to replicate autonomously in the hostcell in question. The origin of replication may be any plasmidreplicator mediating autonomous replication which functions in a cell.The term “origin of replication” or “plasmid replicator” is definedherein as a nucleotide sequence that enables a plasmid or vector toreplicate in vivo.

Examples of origins of replication for use in a yeast host cell are the2 micron origin of replication, ARS1, ARS4, the combination of ARS1 andCEN3, and the combination of ARS4 and CEN6.

Examples of origins of replication useful in a filamentous fungal cellare AMA1 and ANS1 (Gems et al., 1991, Gene 98:61-67; Cullen et al.,1987, Nucleic Acids Research 15: 9163-9175; WO 00/24883). Isolation ofthe AMA1 gene and construction of plasmids or vectors comprising thegene can be accomplished according to the methods disclosed in WO00/24883.

More than one copy of a polynucleotide of the present invention may beinserted into the host cell to increase production of the gene product.An increase in the copy number of the polynucleotide can be obtained byintegrating at least one additional copy of the sequence into the hostcell genome or by including an amplifiable selectable marker gene withthe polynucleotide where cells containing amplified copies of theselectable marker gene, and thereby additional copies of thepolynucleotide, can be selected for by cultivating the cells in thepresence of the appropriate selectable agent.

The procedures used to ligate the elements described above to constructthe recombinant expression vectors of the present invention are wellknown to one skilled in the art (see, e.g., Sambrook et al, 1989,supra).

Host Cells

The present invention also relates to recombinant host cells, comprisinga polynucleotide of the present invention, which are advantageously usedin the recombinant production of the polypeptides. A vector comprising apolynucleotide of the present invention is introduced into a host cellso that the vector is maintained as a chromosomal integrant or as aself-replicating extra-chromosomal vector as described earlier. The term“host cell” encompasses any progeny of a parent cell that is notidentical to the parent cell due to mutations that occur duringreplication. The choice of a host cell will to a large extent dependupon the gene encoding the polypeptide and its source.

The host cell may be a eukaryote, such as a mammalian, insect, plant, orfungal cell.

In a preferred aspect, the host cell is a fungal cell. “Fungi” as usedherein includes the phyla Ascomycota, Basidiomycota, Chytridiomycota,and Zygomycota (as defined by Hawksworth et al., In, Ainsworth andBisby's Dictionary of The Fungi, 8th edition, 1995, CAB International,University Press, Cambridge, UK) as well as the Oomycota (as cited inHawksworth et al., 1995, supra, page 171) and all mitosporic fungi(Hawksworth et al., 1995, supra).

In a more preferred aspect, the fungal host cell is a yeast cell.“Yeast” as used herein includes ascosporogenous yeast (Endomycetales),basidiosporogenous yeast, and yeast belonging to the Fungi Imperfecti(Blastomycetes). Since the classification of yeast may change in thefuture, for the purposes of this invention, yeast shall be defined asdescribed in Biology and Activities of Yeast (Skinner, F. A., Passmore,S. M., and Davenport, R. R., eds, Soc. App. Bacteriol. Symposium SeriesNo. 9, 1980).

In an even more preferred aspect, the yeast host cell is a Candida,Hansenula, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, orYarrowia cell.

In a most preferred aspect, the yeast host cell is a Saccharomycescarlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastatlcus,Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomycesnorbensis, or Saccharomyces oviformis cell. In another most preferredaspect, the yeast host cell is a Kluyveromyces lactis cell. In anothermost preferred aspect, the yeast host cell is a Yarrowia lipolyticacell.

In another more preferred aspect, the fungal host cell is a filamentousfungal cell. “Filamentous fungi” include all filamentous forms of thesubdivision Eumycota and Oomycota (as defined by Hawksworth et al.,1995, supra). The filamentous fungi are generally characterized by amycelial wall composed of chitin, cellulose, glucan, chitosan, mannan,and other complex polysaccharides. Vegetative growth is by hyphalelongation and carbon catabolism is obligately aerobic. In contrast,vegetative growth by yeasts such as Saccharomyces cerevisiae is bybudding of a unicellular thallus and carbon catabolism may befermentative.

In an even more preferred aspect, the filamentous fungal host cell is anAcremonium, Aspergillus, Aureobasidium, Bjerkandera, Ceriporiopsis,Coprinus, Coriolus, Cryptococcus, Filibasidium, Fusarium, Humicola,Magnaporthe, Mucor, Myceliophthora, Neocallimastix, Neurospora,Paecilomyces, Penicillium, Phanerochaete, Phlebia, Piromyces, Pleurotus,Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolypocladium,Trametes, or Trichoderma cell.

In a most preferred aspect, the filamentous fungal host cell is anAspergillus awamori Aspergillus fumigatus, Aspergillus foetidus,Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger orAspergillus oryzae cell. In another most preferred aspect, thefilamentous fungal host cell is a Fusarium bactridioides, Fusariumcerealis, Fusarium crookwellense, Fusarium culmorum, Fusariumgraminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi,Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusariumsambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusariumsulphureum, Fusarium torulosum, Fusarium trichothecioides, or Fusariumvenenatum cell. In another most preferred aspect, the filamentous fungalhost cell is a Bjerkandera adusta, Ceripordopsis subvermispora, Coprinuscinereus, Coriolus hirsutus, Humicola insolens, Humicola lanuginosa,Mucor miehei, Mycellophthora thermophila, Neurospora crassa, Penicilliumpurpurogenum, Phanerochaete chrysosporium, Phlebia radiata, Pleurotuseryngii, Thielavia terrestris, Trametes villosa, Trametes versicolor,Trichoderma harzianum, Trichoderma koningii, Trichodermalongibrachiatum, Trichoderma reesei, or Trichoderma viride strain cell.In an even most preferred aspect, the filamentous fungal strain is aCeriporiopsis aneirina, Ceriporiopsis aneirina, Ceriporiopsis caregiea,Ceriporiopsis gilvescens, Cerlporiopsis pannocinta, Ceriporiopsisrivulosa, Ceriporiopsis subrufa, or Ceriporiopsis subvermispora cell.

Fungal cells may be transformed by a process involving protoplastformation, transformation of the protoplasts, and regeneration of thecell wall in a manner known per se. Suitable procedures fortransformation of Aspergillus and Trichoderma host cells are describedin EP 238 023 and Yelton et al., 1984, Proceedings of the NationalAcademy of Sciences USA 81: 1470-1474. Suitable methods for transformingFusarium species are described by Malardier et al., 1989, Gene 78:147-156, and WO 96/00787. Yeast may be transformed using the proceduresdescribed by Becker and Guarente, In Abelson, J. N. and Simon, M. I.,editors, Guide to Yeast Genetics and Molecular Biology, Methods inEnxymology, Volume 194, pp 182-187, Academic Press, Inc., New York; Itoet al., 1983, Journal of Bacteriology 153: 163; and Hinnen et al., 1978,Proceedings of the National Academy of Sciences USA 75: 1920.Ceriporiopsis may be transformed using the methods for Aspergillus orFusarium described above.

Methods of Production

The present invention also relates to methods for producing apolypeptide of the present invention, comprising (a) cultivating a cell,which in its wild-type form is capable of producing the polypeptide,under conditions conducive for production of the polypeptide; and (b)recovering the polypeptide. Preferably, the cell is of the genusCeriporiopsis, and more preferably Ceriporiopsis subvermispora.

The present invention also relates to methods for producing apolypeptide of the present invention, comprising (a) cultivating a hostcell under conditions conducive for production of the polypeptide; and(b) recovering the polypeptide.

The present invention also relates to methods for producing apolypeptide of the present invention, comprising (a) cultivating a hostcell under conditions conducive for production of the polypeptide,wherein the host cell comprises a mutant nucleotide sequence having atleast one mutation in the polypeptide coding region of SEQ ID NO: 1,wherein the mutant nucleotide sequence encodes a polypeptide whichconsists of SEQ ID NO: 2, and (b) recovering the polypeptide.

In the production methods of the present invention, the cells arecultivated in a nutrient medium suitable for production of thepolypeptide using methods well known in the art. For example, the cellmay be cultivated by shake flask cultivation, and small-scale orlarge-scale fermentation (including continuous, batch, fed-batch, orsolid state fermentations) in laboratory or industrial fermentorsperformed in a suitable medium and under conditions allowing thepolypeptide to be expressed and/or isolated. The cultivation takes placein a suitable nutrient medium comprising carbon and nitrogen sources andinorganic salts, using procedures known in the art. Suitable media areavailable from commercial suppliers or may be prepared according topublished compositions (erg., in catalogues of the American Type CultureCollection). If the polypeptide is secreted into the nutrient medium,the polypeptide can be recovered directly from the medium. If thepolypeptide is not secreted, it can be recovered from cell lysates.

The polypeptides may be detected using methods known in the art that arespecific for the polypeptides. These detection methods may include useof specific antibodies, formation of an enzyme product, or disappearanceof an enzyme substrate. For example, an enzyme assay may be used todetermine the activity of the polypeptide as described herein.

The resulting polypeptide may be recovered using methods known in theart. For example, the polypeptide may be recovered from the nutrientmedium by conventional procedures including, but not limited to,centrifugation, filtration, extraction, spray-drying, evaporation, orprecipitation.

The polypeptides of the present invention may be purified by a varietyof procedures known in the art including, but not limited to,chromatography (e.g., ion exchange, affinity, hydrophobic,chromatofocusing, and size exclusion), electrophoretic procedures (e.g.,preparative isoelectric focusing), differential solubility (e.g.,ammonium sulfate precipitation), SDS-PAGE, or extraction (see, e.g.,Protein Purification, J.-C. Janson and Lars Ryden, editors, VCHPublishers, New York, 1989) to obtain substantially pure polypeptides.

Plants

The present invention also relates to a transgenic plant, plant part, orplant cell which has been transformed with a nucleotide sequenceencoding a polypeptide having oxaloacetate hydrolase activity of thepresent invention so as to express and produce the polypeptide inrecoverable quantities. The polypeptide may be recovered from the plantor plant part. Alternatively, the plant or plant part containing therecombinant polypeptide may be used as such for improving the quality ofa food or feed, e.g., improving nutritional value, palatability, andrheological properties, or to destroy an antinutritive factor.

The transgenic plant can be dicotyledonous (a dicot) or monocotyledonous(a monocot). Examples of monocot plants are grasses, such as meadowgrass (blue grass, Poa), forage grass such as Festuca, Lolium, temperategrass, such as Agrostis, and cereals, e.g., wheat, oats, rye, barley,rice, sorghum, and maize (corn).

Examples of dicot plants are tobacco, legumes, such as lupins, potato,sugar beet, pea, bean and soybean, and cruciferous plants (familyBrassicaceae), such as cauliflower, rape seed, and the closely relatedmodel organism Arabidopsis thaliana.

Examples of plant parts are stem, callus, leaves, root, fruits, seeds,and tubers as well as the individual tissues comprising these parts,e.g., epidermis, mesophyll, parenchyme, vascular tissues, meristems.Specific plant cell compartments; such as chloroplasts, apoplasts,mitochondria, vacuoles, peroxisomes and cytoplasm are also considered tobe a plant part. Furthermore, any plant cell, whatever the tissueorigin, is considered to be a plant part. Likewise, plant parts such asspecific tissues and cells isolated to facilitate the utilisation of theinvention are also considered plant parts, e.g., embryos, endosperms,aleurone and seeds coats.

Also included within the scope of the present invention are the progenyof such plants, plant parts, and plant cells.

The transgenic plant or plant cell expressing a polypeptide of thepresent invention may be constructed in accordance with methods known inthe art. In short, the plant or plant cell is constructed byincorporating one or more expression constructs encoding a polypeptideof the present invention into the plant host genome and propagating theresulting modified plant or plant cell into a transgenic plant or plantcell.

The expression construct is conveniently a nucleic acid construct whichcomprises a polynucleotide encoding a polypeptide of the presentinvention operably linked with appropriate regulatory sequences requiredfor expression of the nucleotide sequence in the plant or plant part ofchoice. Furthermore, the expression construct may comprise a selectablemarker useful for identifying host cells into which the expressionconstruct has been integrated and DNA sequences necessary forintroduction of the construct into the plant in question (the latterdepends on the DNA introduction method to be used).

The choice of regulatory sequences, such as promoter and terminatorsequences and optionally signal or transit sequences is determined, forexample, on the basis of when, where, and how the polypeptide is desiredto be expressed. For instance, the expression of the gene encoding apolypeptide of the present invention may be constitutive or inducible,or may be developmental, stage or tissue specific, and the gene productmay be targeted to a specific tissue or plant part such as seeds orleaves. Regulatory sequences are, for example, described by Tague etal., 1988, Plant Physiology 86: 506.

For constitutive expression, the 35S-CaMV, the maize ubiquitin 1, andthe rice actin 1 promoter may be used (Franck et al., 1980, Cell 21:285-294, Christensen et al., 1992, Plant Mo. Biol. 18: 675-689; Zhang etal., 1991, Plant Cell 3: 1155-1165). Organ-specific promoters may be,for example, a promoter from storage sink tissues such as seeds, potatotubers, and fruits (Edwards and Coruzzi, 1990, Ann. Rev. Genet. 24:275-303), or from metabolic sink tissues such as meristems (Ito et al.,1994, Plant Mol. Biol. 24: 863-878), a seed specific promoter such asthe glutelin, prolamin, globulin, or albumin promoter from rice (Wu etal., 1998, Plant and Cell Physiology 39: 885-889), a Vicia faba promoterfrom the legumin B4 and the unknown seed protein gene from Vicia faba(Conrad et al., 1998, Journal of Plant Physiology 152: 708-711), apromoter from a seed oil body protein (Chen et al., 1998, Plant and CellPhysiology 39: 935-941), the storage protein napA promoter from Brassicanapus, or any other seed specific promoter known in the art, e.g., asdescribed in WO 91/14772. Furthermore, the promoter may be a leafspecific promoter such as the rbcs promoter from rice or tomato (Kyozukaet al., 1993, Plant Physiology 102: 991-1000, the chlorella virusadenine methyltransferase gene promoter (Mitra and Higgins, 1994, PlantMolecular Biology 26: 85-93), or the aldP gene promoter from rice(Kagaya et al., 1995, Molecular and General Genetics 248: 668-674), or awound inducible promoter such as the potato pin2 promoter (Xu et al.,1993, Plant Molecular Biology 22: 573-588). Likewise, the promoter mayinducible by abiotic treatments such as temperature, drought, oralterations in salinity or induced by exogenously applied substancesthat activate the promoter, e.g., ethanol, oestrogens, plant hormonessuch as ethylene, abscisic acid, and gibberellic acid, and heavy metals.

A promoter enhancer element may also be used to achieve higherexpression of a polypeptide of the present invention in the plant. Forinstance, the promoter enhancer element may be an intron which is placedbetween the promoter and the nucleotide sequence encoding a polypeptideof the present invention. For instance, Xu et al., 1993, supra, disclosethe use of the first intron of the rice actin 1 gene to enhanceexpression.

The selectable marker gene and any other parts of the expressionconstruct may be chosen from those available in the art.

The nucleic acid construct is incorporated into the plant genomeaccording to conventional techniques known in the art, includingAgrobacteriummediated transformation, virus-mediated transformation,microinjection, particle bombardment, biolistic transformation, andelectroporation (Gasser et al., 1990, Science 244: 1293; Potrykus, 1990,Bio/Technology8: 535; Shimamoto etal., 1989, Nature 338: 274).

Presently, Agrobacterium tumefaciens-mediated gene transfer is themethod of choice for generating transgenic dicots (for a review, seeHooykas and Schilperoort, 1992, Plant Molecular Biology 19: 15-38) andcan also be used for transforming monocots, although othertransformation methods are often used for these plants. Presently, themethod of choice for generating transgenic monocots is particlebombardment (microscopic gold or tungsten particles coated with thetransforming DNA) of embryonic calli or developing embryos (Christou,1992, Plant Journal 2: 275-281; Shimamoto, 1994, Current OpinionBiotechnology 5: 158-162; Vasil et al., 1992, Bio/Technology 10:667-674). An alternative method for transformation of monocots is basedon protoplast transformation as described by Omirulleh et al., 1993,Plant Molecular Biology 21: 415-428.

Following transformation, the transformants having incorporated theexpression construct are selected and regenerated into whole plantsaccording to methods well-known in the art. Often the transformationprocedure is designed for the selective elimination of selection geneseither during regeneration or in the following generations by using, forexample, co-transformation with two separate T-DNA constructs or sitespecific excision of the selection gene by a specific recombinase.

The present invention also relates to methods for producing apolypeptide of the present invention comprising (a) cultivating atransgenic plant or a plant cell comprising a polynucleotide encoding apolypeptide having oxaloacetate hydrolase activity of the presentinvention under conditions conducive for production of the polypeptide;and (b) recovering the polypeptide.

Removal or Reduction of Oxaloacetate Hydrolase Activity

The present invention also relates to methods for producing a mutant ofa parent cell, which comprises disrupting or deleting a polynucleotidesequence, or a portion thereof, encoding a polypeptide of the presentinvention, which results in the mutant cell producing less of thepolypeptide than the parent cell when cultivated under the sameconditions.

The mutant cell may be constructed by reducing or eliminating expressionof a nucleotide sequence encoding a polypeptide of the present inventionusing methods well known in the art, for example, insertions,disruptions, replacements, or deletions. In a preferred aspect, thenucleotide sequence is inactivated. The nucleotide sequence to bemodified or inactivated may be, for example, the coding region or a partthereof essential for activity, or a regulatory element required for theexpression of the coding region. An example of such a regulatory orcontrol sequence may be a promoter sequence or a functional partthereof, ie., a part that is sufficient for affecting expression of thenucleotide sequence. Other control sequences for possible modificationinclude, but are not limited to, a leader, polyadenylation sequence,propeptide sequence, signal peptide sequence, transcription terminator,and transcriptional activator.

Modification or inactivation of the nucleotide sequence may be performedby subjecting the parent cell to mutagenesis and selecting for mutantcells in which expression of the nucleotide sequence has been reduced oreliminated. The mutagenesis, which may be specific or random, may beperformed, for example, by use of a suitable physical or chemicalmutagenizing agent, by use of a suitable oligonucleotide, or bysubjecting the DNA sequence to PCR generated mutagenesis. Furthermore,the mutagenesis may be performed by use of any combination of thesemutagenizing agents.

Examples of a physical or chemical mutagenizing agent suitable for thepresent purpose include ultraviolet (UV) irradiation, hydroxylamine,N-methyl-N′-nitro-N-nitrosoguanidine (MNNG), O-methyl hydroxylamine,nitrous acid, ethyl methane sulphonate (EMS), sodium bisulphite, formicacid, and nucleotide analogues.

When such agents are used, the mutagenesis is typically performed byincubating the parent cell to be mutagenized in the presence of themutagenizing agent of choice under suitable conditions, and screeningand/or selecting for mutant cells exhibiting reduced or no expression ofthe gene.

Modification or inactivation of the nucleotide sequence may beaccomplished by introduction, substitution, or removal of one or morenucleotides in the gene or a regulatory element required for thetranscription or translation thereof. For example, nucleotides may beinserted or removed so as to result in the introduction of a stop codon,the removal of the start codon, or a change in the open reading frame.Such modification or inactivation may be accomplished by site-directedmutagenesis or PCR generated mutagenesis in accordance with methodsknown in the art. Although, in principle, the modification may beperformed in vivo, i.e., directly on the cell expressing the nucleotidesequence to be modified, it is preferred that the modification beperformed in vitro as exemplified below.

An example of a convenient way to eliminate or reduce expression of anucleotide sequence by a cell is based on techniques of genereplacement, gene deletion, or gene disruption. For example, in the genedisruption method, a nucleic acid sequence corresponding to theendogenous nucleotide sequence is mutagenized in vitro to produce adefective nucleic acid sequence which is then transformed into theparent cell to produce a defective gene. By homologous recombination,the defective nucleic acid sequence replaces the endogenous nucleotidesequence. It may be desirable that the defective nucleotide sequencealso encodes a marker that may be used for selection of transformants inwhich the nucleotide sequence has been modified or destroyed. In aparticularly preferred aspect, the nucleotide sequence is disrupted witha selectable marker such as those described herein.

Alternatively, modification or inactivation of the nucleotide sequencemay be performed by established anti-sense techniques using a sequencecomplementary to the nucleotide sequence. More specifically, expressionof the nucleotide sequence by a cell may be reduced or eliminated byintroducing a sequence complementary to the nucleotide sequence of thegene that may be transcribed in the cell and is capable of hybridizingto the mRNA produced in the cell. Under conditions allowing thecomplementary anti-sense nucleotide sequence to hybridize to the mRNA,the amount of protein translated is thus reduced or eliminated.

The present invention further relates to a mutant cell of a parent cellwhich comprises a disruption or deletion of a nucleotide sequenceencoding the polypeptide or a control sequence thereof, which results inthe mutant cell producing less of the polypeptide or no polypeptidecompared to the parent cell.

The polypeptide-deficient mutant cells so created are particularlyuseful as host cells for the expression of native and/or heterologouspolypeptides. Therefore, the present invention further relates tomethods for producing a homologous or heterologous polypeptidecomprising (a) cultivating the mutant cell under conditions conducivefor production of the polypeptide; and (b) recovering the polypeptide.The term “heterologous polypeptides” is defined herein as polypeptideswhich are not native to the host cell, a native protein in whichmodifications have been made to alter the native sequence, or a nativeprotein whose expression is quantitatively altered as a result of amanipulation of the host cell by recombinant DNA techniques.

In a further aspect, the present invention relates to a method forproducing a protein product essentially free of oxaloacetate hydrolaseactivity by fermentation of a cell which produces both a polypeptide ofthe present invention as well as the protein product of interest byadding an effective amount of an agent capable of inhibitingoxaloacetate hydrolase activity to the fermentation broth before,during, or after the fermentation has been completed, recovering theproduct of interest from the fermentation broth, and optionallysubjecting the recovered product to further purification.

In a further aspect, the present invention relates to a method forproducing a protein product essentially free of oxaloacetate hydrolaseactivity by cultivating the cell under conditions permitting theexpression of the product, subjecting the resultant culture broth to acombined pH and temperature treatment so as to reduce the oxaloacetatehydrolase activity substantially, and recovering the product from theculture broth. Alternatively, the combined pH and temperature treatmentmay be performed on an enzyme preparation recovered from the culturebroth. The combined pH and temperature treatment may optionally be usedin combination with a treatment with a oxaloacetate hydrolase inhibitor.

In accordance with this aspect of the invention, it is possible toremove at least 60%, preferably at least 75%, more preferably at least85%, still more preferably at least 95%, and most preferably at least99% of the oxaloacetate hydrolase activity. Complete removal ofoxaloacetate hydrolase activity may be obtained by use of this method.

The combined pH and temperature treatment is preferably carried out at apH in the range of 8-9 and a temperature in the range of 65-70° C. for asufficient period of time to attain the desired effect, where typically,30 to 60 minutes is sufficient.

The methods used for cultivation and purification of the product ofinterest may be performed by methods known in the art.

The methods of the present invention for producing an essentiallyoxaloacetate hydrolase-free product is of particular interest in theproduction of eukaryotic polypeptides, in particular fungal proteinssuch as enzymes. The enzyme may be selected from, e.g., an amylolyticenzyme, lipolytic enzyme, proteolytic enzyme, cellulytic enzyme,oxidoreductase, or plant cell-wall degrading enzyme. Examples of suchenzymes include an aminopeptidase, amylase, amyloglucosidase,carbohydrase, carboxypeptidase, catalase, cellulase, chitinase,cutinase, cyclodextrin glycosyltransferase, deoxyribonuclease, esterase,galactosidase, beta-galactosidase, glucoamylase, glucose oxidase,glucosidase, haloperoxidase, hemicellulase, invertase, isomerase,laccase, ligase, lipase, lyase, mannosidase, oxidase, pectinolyticenzyme, peroxidase, phytase, phenoloxidase, polyphenoloxidase,proteolytic enzyme, ribonuclease, transferase, transglutaminase, orxylanase. The oxaloacetate hydrolase-deficient cells may also be used toexpress heterologous proteins of pharmaceutical interest such ashormones, growth factors, receptors, and the like.

It will be understood that the term “eukaryotic polypeptides” includesnot only native polypeptides, but also those polypeptides, e.g.,enzymes, which have been modified by amino acid substitutions, deletionsor additions, or other such modifications. to enhance activity,thermostability, pH tolerance and the like.

In a further aspect, the present invention relates to a protein productessentially free from oxaloacetate hydrolase activity which is producedby a method of the present invention.

Compositions

The present invention also relates to compositions comprising apolypeptide of the present invention. Preferably, the compositions areenriched in such a polypeptide. The term “enriched” indicates that theoxaloacetate hydrolase activity of the composition has been increased,e.g., with an enrichment factor of 1.1.

The composition may comprise a polypeptide of the present invention asthe major enzymatic component, e.g., a mono-component composition.Alternatively, the composition may comprise multiple enzymaticactivities, such as an aminopeptidase, amylase, carbohydrase,carboxypeptidase, catalase, cellulase, chitinase, cutinase, cyclodextringlycosyltransferase, deoxyribonuclease, esterase, alpha-galactosidase,beta-galactosidase, glucoamylase, alpha-glucosidase, beta-glucosidase,haloperoxidase, invertase, laccase, lipase, mannosidase, oxidase,pectinolytic enzyme, peptidoglutaminase, peroxidase, phytase,polyphenoloxidase, proteolytic enzyme, ribonuclease, transglutaminase,or xylanase. The additional enzyme(s) may be produced, for example, by amicroorganism belonging to the genus Aspergillus, preferably Aspergillusaculeatus, Aspergillus awamori, Aspergillus fumigatus, Aspergillusfoetidus, Aspergillus japonicus, Aspergillus nidulans, Aspergillusniger, or Aspergillus oryzae, Fusarium, preferably Fusariumbactridioides, Fusarium cerealis, Fusarium crookwellense, Fusariumculmorum, Fusarium graminearum, Fusardum graminum, Fusariumheterosporum, Fusardum negundi, Fusarium oxysporum, Fusariumreticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum,Fusarium sulphureum, Fusarium toruloseum, Fusarium trichothecioides, orFusarlum venenatum, Humicola, preferably Humicola insolens or Humicolalanuginosa; or Trichoderma, preferably Trichoderma harzianum,Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei,or Trichoderma viride.

The polypeptide compositions may be prepared in accordance with methodsknown in the art and may be in the form of a liquid or a drycomposition. For instance, the polypeptide composition may be in theform of a granulate or a microgranulate. The polypeptide to be includedin the composition may be stabilized in accordance with methods known inthe art.

Examples are given below of preferred uses of the polypeptidecompositions of the invention. The dosage of the polypeptide compositionof the invention and other conditions under which the composition isused may be determined on the basis of methods known in the art.

Uses

The present invention is also directed to methods for using thepolypeptides having oxaloacetate hydrolase activity.

The polypeptides of the present invention may be used as a diagnosticenzyme, e.g., for the detection of oxalic acid in food or otherproducts.

The nucleotide sequences of the present invention may be used formodification of the production of oxaloacetate hydrolase and thus oxalicacid by a cell, such as a microbial cell normally producing thehydrolase. In particular, the nucleotide sequences may be used to reduceor eliminate oxaloacetate hydrolase and thus oxalic acid production ofthe cell in question.

Strains may be constructed, as described herein, to overproduce apolypeptide of the present invention to improve the lignin degradativeability of the strain. In a preferred aspect, the strain is a white rotfungus known to be involved in the degradation of lignin. In a morepreferred aspect, the strain is Ceriporiopsis strain. In an even morepreferred aspect, the strain is Ceriporiopsis aneirina, Ceriporiopsisaneirina, Ceriporiopsis caregiea, Ceriporiopsis gilvescens,Ceriporiopsis pannocinta, Ceriporiopsis rivulosa, Ceriporiopsis subrufa,or Ceriporiopsis subvermispora strain. In a most preferred aspect, thestrain is Ceriporiopsis subvermispora, and most preferably Ceriporiopsissubvermispora FPL 104807SS-5. In another more preferred aspect, thestrain is a Bjerkandera, Coprinus, Coriolus, Humicola, Phanerochaete,Phlebia, Pleurotus, Thielavia, Trametes, or Trichoderma strain. Inanother most preferred aspect, the strain is a Bjerkandera adusta,Coprinus cinereus, Coriolus hirsutus, Humicola insolens, Humicolalanuginosa, Phanerochaete chrysospordum, Phlebia radiata, Pleurotuseryngii, Thielavia terrestris, Trametes villosa, Trametes versicolor,Trichoderma harzianum, Trdchoderma koningii, Trichodermalongibrachiatum, Trichoderma reesei, or Trichoderma viride strain.

The improved strains may then be used for the colonization oflignin-containing material, for example, wood, using the proceduresdescribed by Messner and Srebotnik, 1994, FEMS Microb. Rev. 13: 351-364;Scott and Swaney, 1998, TAPPIJ, 81: 153-175; and Breen and Singleton,1999, Curr. Opinion Biotech. 10: 252-258.

The present invention is further described by the following exampleswhich should not be construed as limiting the scope of the invention.

EXAMPLES

Chemicals used as buffers and substrates were commercial products of atleast reagent grade.

Media and Solution PDA plates were composed per liter of 39 grams ofpotato dextrose agar.

YEG was composed per liter of 5 g of yeast extract and 20 g of glucose.

LB medium (pH 7.4) was composed per liter of 10 g of bacto-tryptone, 5 gof yeast extract, and 10 g of NaCI pH 7.4.

2×LB medium (pH 7.4) was composed per liter of 20 g of bacto-tryptone,10 g of yeast extract, and 20 g of NaCl pH 7.4.

Basal minimal medium was composed per liter of 10 g of glucose, 1 mMammonium tartrate, 10 mM transaconitic acid (pH 4.5), 2 g of KH₂PO₄, 0.5g of MgSO₄.7H₂O, 0.1 g of CaCl₂.2H₂O, 1 mg of thiamine hydrochloride,and 1 ml of trace elements solution.

Trace elements solution was composed per liter of 15 g of nitroaceticacid, 1 g of FeSO₄.7H₂O, 1.8 g of CoCl₂.6H₂O, 1 g of ZnCl₂.7H₂O, 0.07 gof Al₂(SO₄)₃.18H₂O, 0.1 g of CuSO₄.5H₂O, 0.1 g of H₃BO₃, 0.1 g ofNaMoO₄.2H₂O, 30 g of MgSO₄.7H₂O, 10 g of NaCl, 0.82 g of CaCl₂, and 0.5g of MnSO₄.

2×YT plates were composed per liter of 15 g of Bacto Tryptone, 10 g ofyeast extract, and 5 g of NaCl.

SOC medium was composed per liter of 20 g of Bacto Tryptone, 5 g ofyeast extract, 2 ml of 5 M NaCl, and 2.5 ml of 1M KCl.

Example 1 Isolation of Genomic DNA from Ceriporiopsis Subvermispora

A quarter inch square of agar of Ceriporiopsis subvermispora FPL104807SS-5 (Forest Products Laboratory, Madison, Wis.) mycelia from aPDA plate was inoculated into 250 ml of YEG medium and incubated at 28°C. for 5 days. The mycelia were harvested by filtration throughMiracloth (Calbiochem, San Diego, Calif.) and were frozen quickly inliquid nitrogen. Genomic DNA was isolated according to the procedure ofWalheitner et al., 1996, Current Genetics 29: 395-403. The DNA pelletwas resuspended in 500 μl of TE buffer (10 mM Tris-1 mM EDTA), and 5 μlwas electrophoresed on a 1% agarose gel using TAE buffer (4.84 g of TrisBase, 1.14 ml of glacial acetic acid, and 2 ml of 0.5 M EDTA pH 8.0 perliter) to confirm the quality of the preparation.

Example 2 Construction of a Ceriporiopsis Subvermispora Genomic Library

A total of 30 μg of Ceriporiopsis subvermispora genomic DNA was digestedwith Tsp509I (New England Biolabs, Beverly, Mass.) in a total volume of150 μl using the manufacturer's recommended conditions. Aliquots of 30μl of the digested DNA were removed at 10, 11, 12, 14, and 16 minutesafter the addition of Tsp509I. The digestions were electrophoresed on a0.8% agarose gel using TAE buffer. A gel slice containing 2 to 3 kb ofdigested DNA was removed, and the DNA fragments were purified usingβ-agarase (New England Biolabs, Beverly, Mass.) following themanufacturer's protocols.

A total of 3 μg of pUC19 was digested with EcoRI and treated with shrimpalkaline phosphatase (Amersham Pharmacia Biotech, Arlington Heights,Ill.) following the manufacturer's protocols. The digested DNA waselectrophoresed on a 0.8% agarose gel using TAE buffer, and the gelslice containing the linearized plasmid was excised from the gel. Theplasmid DNA was isolated from the gel slice using a Qiaquick spin column(QIAGEN, Chatsworth, Calif.).

The 2-3 kb Tsp509l genomic fragments and the EcoRI digested pUC19 wereligated together overnight at room temperature with T4 DNA ligase (NewEngland Biolabs, Beverly, Mass.). The ligation reaction was precipitatedby adding 1/10 volume of 3 M sodium acetate pH 5.0 and 2.5 volumes of95% ethanol, incubating on ice for 30 minutes, and centrifuging at12,000×g for 30 minutes. The ligated DNA was resuspended in 10 μl of TEbuffer.

Three 40 μl aliquots of E. coli Electromax DH10B competent cells (GibcoBRL, Bethesda, Md.) were transformed with 1 μl of the ligated DNA byelectroporation at a time constant of 2.3 at 2.5 kV, 25 μF, 100 Ω in a0.1 cm cuvette. After transformation the cells were centrifuged at1660×g for 5 to 10 minutes. The supernatant was carefully removed andthe pelleted cells were resuspended in 0.5 ml of 2×LB medium plus 0.5 mlof 50% sterile glycerol. The transformants were frozen quickly in a dryice ethanol bath and then frozen at −80° C.

The three transformations were titered after 1 day at −80° C. by platingon LB plates supplemented with 100 μg of ampicillin per ml. The totalnumber of independent transformants obtained were approximately 23,000,32,500, and 34,500 per transformation. The frozen glycerol stocks weresent to Genome Systems (St. Louis, Mo.) for colony picking. A library ofapproximately 50,000 in 384-well plates was obtained.

Example 3 Isolation of RNA from Ceriporiopsis Subvermispora

Six 1 liter flasks with 29 ml of basal minimal medium were incubatedwith agar plugs from a PDA plate containing Ceriporiopsis subvermisporamycelia. The flasks were incubated at 28° C. without shaking for 15days. The mycelia mats were harvested from the flasks and homogenized insterile water in a Waring blender three times for 15 seconds with 30second intervals to prevent the mycelia from warming. The homogenizedmycelia were added to a 1 liter flask containing 20 μm of hardwoodthermomechanical pulp that had previously been processed in a Waringblender for 30 seconds, and the mixture was stirred. For the minimalmedium cultures, the homogenized mycelia were added to 15 ml of minimalmedium in 1 liter flasks. The cultures were incubated at 30° C. for 30days without shaking and the entire pulp culture containing pulp plusmycelia was frozen quickly in liquid nitrogen. The mycelia from theminimal medium culture were harvested by filtration through Miraclothand quickly frozen in liquid nitrogen.

RNA was prepared from the culture using a phenol/chloroform extractionas described below. Fresh p-aminosalicylic (PAS) (Sigma Chemical Co.,St. Louis, Mo.) solution was prepared by mixing 9.6 μm in 80 ml ofdiethylpyrocarbonate (DEP)-treated water (Amresco, Solon, Ohio). Freshtri-isopropyinaphthalene sulfonic acid (TNS) solution was prepared bymixing 1.6 gm in 80 ml of DEP-treated water. A 5×RNB solution wasprepared by adding 24.2 g of Tris-HCl, 14.6 g of NaCl and 19 g of EGTAto 200 ml of DEP-treated water and adjusting the pH to 8.5 with NaOH.RNA extraction buffer was prepared by adding the PAS solution to the TNSsolution while stirring. The PAS/TNS mixture was then added to 40 ml ofRNB while stirring, and the final solution was placed on ice.

The frozen mycelia or mycelia plus pulp were ground to a fine powder ina coffee grinder that was prechilled with a few chips of dry ice. Thepowder was immediately added to 20 ml of RNA extraction buffer followedby 0.5 volumes of phenol/chloroform (1:1 v/v), and the mixture wasplaced on ice. A 0.25 volume of phenol/chloroform (1:1 v/v) was addedand the phases were separated by centrifugation at 800×g for 10 minutes.The aqueous phase was removed and placed on ice in a fresh 50 ml tubecontaining a few drops of phenol/chloroform (1:1 v/v). The organic phasewas mixed with 2 ml of extraction buffer, incubated in a water bath at68° C. for 5 minutes, and centrifuged as above. The aqueous phase wascombined with that saved on ice. The aqueous phase was then extractedfour times with phenol/chloroform (1:1 v/v) until there was no proteinat the interface. To precipitate the RNA, 0.1 volume of 3 M sodiumacetate pH 5.2 plus 2.5 volumes of 95% ethanol was added and the mixturewas frozen at −20° C. for 2 hours. The RNA was pelleted bycentrifugation at 12,000×g for 20 minutes and resuspended in 450 μl ofDEP-treated water. A 2 μl aliquot of each total RNA preparation waselectrophoresed on a 0.8% agarose gel using TAE buffer to check thequality of the RNA.

Poly-A RNA was isolated using an mRNA Separator Kit (Clonetech, PaloAlto, Calif.) according to the manufacturer's protocols.

Example 4 Isolation of Plasmid DNA from the Genomic Library Clones

Each 384 well plate containing genomic clones was used to inoculate four96-well deep well plates containing 1.25 ml of Magnificent Broth(MaCconnell Research, San Diego, Calif.) supplemented with ampicillin at50 μg per ml. The 96-well deep well plates were incubated at 37° C. for22-24 hours at 300 rpm. The plates were then centrifuged at 800×g for 10minutes. Plasmid DNA was isolated using a Qiaprep Turbo Core Kit(QIAGEN, Chatsworth, Calif.) and a Qiagen BioRobot 9600 (QIAGEN,Chatsworth, Calif.) according to the manufacturer's instructions. The96-well plates containing the plasmid DNA were dried down in a SpeedVac(Savant Instruments, Inc., Holbrook, N.Y.) followed by the addition of15 μl of 3×SSC to each well using a Hydra HTS workstation (RobbinsScientific, Sunnyvale, Calif.).

Example 5 Printing of DNA Microarrays

Four 96-well plates were rearrayed back to 384-well plates using aRobbins Hydra HTS workstation. A 5 μl volume of each plasmid wasaliquoted into 384-well microplates. From these plates, the plasmidswere spotted onto poly-L-lysine coated glass microscope slides using theequipment and methods described in U.S. Pat. No. 5,807,522. The densityof spots was 10,000 per slide.

Example 6 Probe Preparation and Hybridization

Fluorescent probes were prepared by reverse transcription of 1 μg ofpolyA RNA from Ceriporiopsis subvermispora to incorporateaminoallyl-dUTP into first strand cDNA. The amino-cDNA products weresubsequently labeled by direct coupling to either Cy3 or Cy5monofunctional reactive dyes (Amersham Pharmacia Biotech, ArlingtonHeights, Ill.). The details of this protocol are described by DeRisi etal., 1997, Science 278: 680-686. Cy3 and Cy5 labeled probes werecombined and purified using Qiaquick PCR spin columns (QIAGEN, Valencia,Calif.). The purified probes were dried under vacuum in a SpeedVac,resuspended in 18 μl of water, and combined with the following: 3.6 μlof 20×SSC, 1.8 μl of poly-dA (500 μg/ml; Amersham Pharmacia Biotech),and 0.54 μl of 10% SDS. Before hybridization, the solution was filteredwith a 0.22 μm Ultrafree-MC microcentrifuge filter (Millipore, Beford,Mass.), boiled for 2 minutes, and cooled to room temperature. The probewas then applied to the microarray under a coverglass, placed in ahumidified chamber, and incubated at 65° C. overnight. Before scanning,the arrays were washed consecutively in 1×SSC with 0.03% SDS, 0.2×SSC,and 0.05×SSC, and centrifuged for 2 minutes at 500 rpm to remove excessliquid. Finally, the slides were imaged using a custom-built confocallaser microscope (Eisen and Brown, 1999, Methods in Enzymology 303:179).

Example 7 Characterization of Clones Induced by Pulp

From the first 20,000 genomic clones (Example 2), 20 clones weredetermined to contain DNA whose expression was induced on hardwoodthermomechanical pulp based on hybridization with the probe as describedin Example 6. For each of the clones, plasmid DNA was isolated byinoculating 1 μl from the well of the 384 plate containing the desiredclone into a 15 ml Falcon tube containing 3 ml of LB supplemented with100 μg of ampicillin per ml. The clones were grown overnight at 37° C.,250 rpm.

Plasmids were then isolated using the QIAGEN robot protocol described inExample 5 and sequenced using 150 ng of plasmid template, 1.6 ng of M13primer (forward or reverse), and water to 6 μl. The samples were run onan ABI 3700 Sequencer (Applied Biosystems, Foster City, Calif.). One ofthe clones, pCsubHP4 contained a genomic fragment, which shared identityto known oxaloacetate hydrolases but did not contain the whole gene.

Example 8 Utilizing RLM-RACE to Amplify the Complete Coding Sequence ofa Oxaloacetate Hydrolase

Total RNA was prepared according to the method outlined in Example 3.All of the steps in a RLM-RACE reaction were performed using a GeneRacerKit (Invitrogen, Carlsbad, Calif.) according to the manufacturer'smethodology. The RNA was subjected to a dephosphorylation reaction using14 μl of total RNA (about 1 μg), 2 μl of CIP buffer (Invitrogen,Carlsbad, Calif.), 2 μl of RNaseOut (40 U/μl, Invitrogen, Carlsbad,Calif.), and 2 μl of calf intestinal phosphatase (10 U/μl). The reactionwas mixed by pipette, vortexed briefly, and incubated at 50° C. for 1hour. After the incubation the reactions were briefly centrifuged in amicrocentrifuge and placed on ice.

The RNA was then precipitated using the following protocol. Volumes of90 μl of DEPC water and 100 μl of phenol:chloroform 1:1 v/v were addedto the samples and vortexed vigorously for 30 seconds. The samples werethen centrifuged at maximum speed in a microcentrifuge for 5 minutes atroom temperature. The aqueous (top) phase was transferred to a newmicrocentrifuge tube followed by 2 μl of mussel glycogen (10 mg/ml,Invitrogen, Carlsbad, Calif.), and 10 μl of 3 M sodium acetate pH 5.2,mixed well, and frozen on dry ice for 10 minutes. The RNA wassubsequently pelleted by centrifugation at maximum speed in amicrocentrifuge for 20 minutes at 4° C. The supernatant was removed bypipette being careful not to disturb the pellet. A 500 μl volume of 70%ethanol was then added to the pellet, inverted several times, vortexedbriefly, and centrifuged at maximum velocity for 2 minutes at 4° C. in amicrocentrifuge. The ethanol was removed by pipette and recentrifuged,and the step repeated to remove the final traces of ethanol remaining.The RNA pellet was air dried for 2 minutes at room temperature and thenresuspended in 7 μl of DEPC water.

The mRNA cap structure was then removed with 7 μl of thedephosphorylated RNA, 1 μl of 10×tobacco acid pyrophosphatase buffer(Invitrogen, Carlsbad, Calif.), 1 μl of RNaseOut (40 U/μl), 1 μl of TAP(0.5 U/μl, Invitrogen, Carlsbad, Calif.), which was mixed by pipette andvortexed briefly. The fluid was collected by a short centrifuge pulse ina microcentrifuge and subsequently incubated at 37° C. for 1 hour. Afterincubation the samples were quickly centrifuged and placed on ice. TheRNA was then precipitated as outlined above.

Once the mRNA cap structure was removed, ligation of a GeneRacer RNAoligo was performed by reacting 7 μl of dephosphorylated, decapped RNAwith 0.25 μg of the GeneRacer RNA oligo shown below:5′-CGACUGGAGCACGAGGACACUGACAUGGACUGA (SEQ ID NO: 3) AGGAGUAGAAA-3′

The mixture was pipetted several times and then incubated at 65° C. for5 minutes to remove the RNA secondary structure. After incubation thereaction was chilled on ice for 2 minutes followed by a briefcentrifugation. To this mixture, 1 μl of 10×ligase buffer, 1 μl of 10 mMATP, 1 μl of RNaseOut (40 U/μl), and 1 pi of T4 RNA ligase (5 U/μl) wereadded and incubated for 1 hour at 37° C. The reaction was thencentrifuged briefly and placed on ice after which the RNA wasprecipitated up to the dry ice freezing step according to theprecipitation protocol and afterward stored at −20° C. for the night.

The precipitation protocol was completed the next morning prior to themRNA reverse transcription. The reverse transcription was carried out in3 different reactions each with a different gene specific reverseprimer. The primers were constructed to amplify 3′-ends of a partialgene fragment which appeared to be induced during growth ofCeriporiopsis subvermispora on pulp based on the procedure described inExample 3. The sequence was based upon the consensus sequence of thepositive array clones. The different gene specific primers were asfollows:

-   -   (1) 5′-GCCACCTGCTTGGCAAGCAGGTCGTCT-3′ (Oligo ID# 993605 for the        reverse transcription of the CsubHP4 gene encoding a        oxaloacetate hydrolase) (SEQ ID NO: 4)    -   (2) 5′-ACCGGCTTCGGTGGTCCCGCTATGGTT-3′ (Oligo ID# 993606 for the        reverse transcription of the CsubHP4 gene encoding a        oxaloacetate hydrolase. Located at a different position than        (1)) (SEQ ID NO: 5)    -   (3) 5′-GAGCGCACAGATGGTCTGCAGCTTGM-3′ (Oligo ID# 993607 for the        reverse transcription of the CsubHP4 gene encoding a        oxaloacetate hydrolase. Located at a different position than (1)        and (2)) (SEQ ID NO: 6)

Volumes of 1 μl of the oligo dT primer (from the Gene Racer Kit) and 1μl of dNTP mix (10 mM each) were added to 10 μl of the ligated RNA. Thesolution was incubated at 65° C. to remove any RNA secondary structureand then chilled on ice for 2 minutes. Next, 4 μl of 5×first strandbuffer (Invitrogen, Carlsbad, Calif.), 2 μl of 0.1 M DTT, 1 μl ofRNaseOut (40 U/μl), and 1 μl of Superscript II RT (200 U/μl, Invitrogen,Carlsbad, Calif.) were added to the primer and dNTP's, mixed,centrifuged briefly, and incubated at 42° C. for 50 minutes. After theincubation, the RT reaction was inactivated by heating to 70° C. for 15minutes. The inactivated reaction was chilled on ice for 2 minutes andcentrifuged briefly. A 1 μl volume of RNase H (2 units, Invitrogen,Carlsbad, Calif.) was added to the reaction mixture, incubated for 37°C. for 20 minutes, and then centrifuged briefly.

Once the total dT cDNA was synthesized, the sequence of interest wasamplified by PCR amplification. Three amplification reactions (50 μl)were carried out using primers (1)-(3) above in combination with theGeneRacer dT oligo primer of SEQ ID NO: 3. The reaction mixturecontained 3 μl of GeneRacer dT oligo primer (10 μM), 1 μl of reversegene specific primer (10 μM), 2 μl RT template from the appropriatereaction, 5 μl of 10×Taq buffer (New England Biolabs, Beverly, Mass.), 1μl of dNTP mix (10 mM each), 2.5 μl of DMSO, 34 μl of water, and 0.5 μlof Taq DNA polymerase (New England Biolabs, Beverly, Mass.). Thereaction was incubated in a Eppendorf Mastercycler thermocycler(Eppendorf Scientific, Inc., Westbury, N.Y.), to run a touchdown PCR,programmed for 1 cycle at 94° C. for 2 minutes; 5 cycles each at 94° C.for 30 seconds and then 72° C. for 2 minutes; 5 cycles each at 94° C.for 30 seconds and then 70° C. for 2 minutes; 20 cycles each at 94° C.for 30 seconds, 65° C. for 30 seconds, and 68° C. for 2 minutes; 1 cycleat 68° C. for 10 minutes; and a 4° C. hold overnight.

A 10 μl volume of each PCR was mixed with 1.1 μl of loading buffer andrun on a 0.8% agarose gel using TBE buffer (50 mM Tris base-50 mM boricacid-1 mM disodium EDTA) containing ethidium bromide at 90 V for 1 hour.The products were observed with UV light on a Nucleotech gelvisualization system (Nucleotech, San Mateo, Calif.). The PCRamplifications providing products of approximately 900 bp were subjectedto a round of nested PCR in order to increase concentration and purity.Nested PCR was carried out with the GeneRacer dT oligo primer and one ofthe gene specific primers as described above. The reactions were rununder the same PCR conditions as described above except differenttemplates and nested primers were used as described below:

-   -   (1) GeneRacer dT oligo and Primer (2) PCR product as template (2        μl) with the GeneRacer dT oligo primer and primer (1)    -   (2) GeneRacer dT oligo and Primer (3) PCR product as template (2        μl) with GeneRacer dT oligo primer and primer (1)    -   (3) GeneRacer dT oligo and Primer (3) PCR product as template (2        μl) with GeneRacer dT oligo primer and primer (2)

The PCR products were analyzed by agarose gel electrophoresis accordingto the first PCR reaction protocol described above. Productsapproximately 900 bp in size were ligated into a TOPO-TA vector(Invitrogen, Carlsbad, Calif.) according to the manufacturer'sinstructions by mixing 4 μl of the PCR product, 1 μl of salt solution(Invitrogen, Carlsbad, Calif.), and 1 μl of the TOPO-TA vector with apipette and incubating at room temperature for 30 minutes.

After the incubation, OneShot competent E. coli cells (Invitrogen,Carlsbad, Calif.) were transformed with 2 μl of the ligation mixture andincubated on ice for 5 minutes. Subsequently the cells were heat shockedfor 30 seconds at 42° C. and then placed on ice for 2 minutes. A 250 μlvolume of SOC medium was added to the cells and the mixture wasincubated for 1 hour at 37° C. and 250 rpm. After the incubationcolonies were spread on 2×YT plates supplemented with 100 μg ofampicillin per ml and incubated at 37° C. overnight for selection of theplasmid. Colonies that grew on the plates were picked with a steriletoothpick, transferred to a 15 ml Falcon tube containing 3 ml of LBmedium supplemented with 100 μg of ampicillin per ml, and incubatedovernight at 37° C., 250 rpm.

Plasmids were isolated using the QIAGEN robot protocol described inExample 5. A 2 μl volume of the resulting plasmid minipreps Weredigested with EcoRI. The digestion reactions were analyzed by agaroseelectrophoresis as previously outlined for the PCR reactions to confirmthe quality of the preparation.

Isolated plasmids containing an insert of approximately 900 bp weresequenced using 150 ng of plasmid template, 1.6 ng of M13 primer(forward or reverse), and 6 μl of water. The plasmid samples weresequenced using an ABI 3700 Sequencer (Applied Biosystems, Foster City,Calif.) and the sequences analyzed using Sequencher tools (Genecodes,Ann Arbor, Mich.) and blast alignments (NCBI, Bethesda, Md.) allowingfor identification of an open reading frame for the oxaloacetatehydrolase gene.

Example 9 Utilizing RLM-RACE to Amplify the 5′ end of the CDNA for theOxaloacetate Hydrolase

A RLM-RACE was performed to obtain the 5′ cDNA sequence of theCeriporiopsis subvermispora oxaloactetate hydrolase gene.

Total RNA from hardwood thermomechanical pulp treated for 30 days withCeriporiopsis subvermispora was prepared according to the methodoutlined in Example 3. All of the steps in the RLM-RACE reaction wereperformed with materials provided in the GeneRacer Kit and carried outaccording to the manufacturer's methodology. The RNA was subjected to adephosphorylation reaction using 5 μl of total RNA (about 1 μg), 2 μlCIP buffer, 2 μl RNaseOut (40 U/μl), and 2 μl CIP (10 U/μl). Thereaction was mixed by pipette and vortexed briefly after which it wasincubated at 50° C. for 1 hour. After incubation the reactions werebriefly centrifuged and placed on ice.

The RNA was then precipitated and mRNA cap structure removed asdescribed in Example 8. Once the mRNA cap structure was removed,ligation of the GeneRacer RNA oligo was performed as described inExample 8. The ligation mixture was stored at −20° C. overnight.

The frozen RNA ligation mixture was thawed and subsequently pelleted bycentrifugation at maximum speed in a microcentrifuge for 20 minutes at4° C. The supernatant was removed by pipetting being careful not todisturb the pellet. A 500 μl volume of 70% ethanol was then added to thepellet, inverted several times, vortexed briefly, and then centrifugedat maximum velocity for 2 minutes at 4° C. in a microcentrifuge. Theethanol was removed by pipette and the RNA pellet air dried for 2minutes at room temperature and then resuspended in 7 μl of DEPC water.

Reverse transcription was performed using Random Primers (N₆)(Invitrogen, Inc., Carlsbad, Calif.). A 1 μl volume of random primer mixand 1 μl of dNTP mix (10 mM each) were added to the ligated RNA (10 μl).The mixture was incubated at 65° C. to remove any RNA secondarystructure and then chilled on ice for 2 minutes. Then 4 μl of 5×firststrand buffer (Invitrogen, Inc., Carlsbad, Calif.), 2 μl of 0.1 M DTT, 1μl of RNaseOut (40 U/μl), and 1 μl of 15 U/μl Cloned AMV RT (Invitrogen,Inc., Carlsbad, Calif.) were added to the primer mix and dNTP's, mixedwell, centrifuged briefly, and incubated at room temperature for 10minutes to allow efficient binding of the Random Primer to the template.Immediately after the room temperature incubation the reaction mix wasmoved to a 42° C. water bath for 1 hour. After incubation, the reversetranscription reaction was inactivated by heating to 85° C. for 15minutes, chilling on ice for 2 minutes, and centrifuging briefly.

Once the cDNA was synthesized, the fragment of interest was amplified byPCR using the Expand High Fidelity PCR system (Roche Diagnostics,Mannheim, Germany) with oligo 995222 in combination with the GeneRacer5′ primer shown below. Oligo 995222: 5′-GGACTTCAGGAGCTCGGCGGT-3′ (SEQ IDNO: 7) GeneRacer 5′ primer: 5′-CGACTGGAGCACGAGGACACTGA-3′ (SEQ ID NO: 8)

The reaction mixture (50 μl) contained 1 μl of GeneRacer 5′ primer (10μM), 1 μl of reverse gene specific primer (50 pmol/μl), 5 μl of 10×PCRbuffer with 15 mM MgCl₂, 1 μl of dNTP mix (10 mM each), 40.25 μl water,and 0.75 μl (3.5 U/μl) of DNA polymerase mix. The reaction was incubatedin a Eppendorf Mastercycler thermocycler programmed for 1 cycle at 94°C. for 2 minutes; 10 cycles each at 94° C. for 15 seconds 50° C. for 30seconds, 72° C. for 1.5 minutes; 15 cycles each at 94° C. for 15seconds, 55° C. for 30 seconds, 72° C. for 1.5 minutes plus 5 secondelongation at each successive cycle; 1 cycle at 72° C. for 7 minutes;and a 4° C. hold overnight.

Ten microliters of the PCR reaction was mixed with 1 μl of 10×DNAloading dye (25% glycerol, 10 mM Tris pH 7.0, 10 mM EDTA, 0.025%bromophenol blue, 0.025% xylene cyanol) and run on a 1.0% (w/v) agarosegel using TAE buffer. The products were observed with UV light on aNucleotech gel visualization system. The PCR product was directlyligated into the pPCR4-TOPO vector (Invitrogen, Carlsbad, Calif.)according to the manufacturer's instructions. A 1 μl aliquot of freshPCR product, 3 μl of double distilled water, and 1 μl of the TOPOcloning vector were mixed with a pipette and incubated at roomtemperature for 5 minutes.

After the incubation, OneShot competent E. coli cells were transformedwith 2 μl of the ligation mixture and incubated on ice for 5 minutes.Subsequently the cells were heat shocked for 30 seconds at 42° C. andthen placed on ice for 2 minutes. A 250 μl volume of SOC medium wasadded to the cells and the mixture was incubated for 1 hour at 37° C.and 250 rpm. After the incubation colonies were spread on 2×YT platessupplemented with 100 μg of ampicillin per ml and incubated at 37° C.overnight for selection of the plasmid.

Eight colonies that grew on the 2×YT plates were picked with a steriletoothpick, transferred to a 15 ml Falcon tube containing 3 ml of LBmedium supplemented with 100 μg of ampicillin per ml, and incubatedovernight at 37° C., 250 rpm. The plasmids were isolated using theQIAGEN robot protocol described in Example 5. A 4 μl volume of theresulting plasmid minipreps were digested with EcoRI. The digestionreactions were analyzed by agarose gel electrophoresis as previouslydescribed for the PCR reaction.

Isolated plasmids containing inserts were sequenced using 1 μl ofplasmid template, 1.6 ng of M13 primer (forward or reverse), and 6 μl ofwater. DNA sequencing was performed with an Applied Biosystems Model 377Sequencer XL using dye-terminator chemistry. From the sequenceinformation, the 5′ coding sequence of the Ceriporiopsis subvermisporaoxaloactetate hydrolase gene was deduced.

Example 10 PCR Amplification of 3′ Oxaloacetate Hydrolase Genomic Region

Primers were designed based on the 3′ cDNA sequence for the oxaloacetatehydrolase gene from Ceriporiopsis subvermispora. The primers, shownbelow, were used to PCR amplify the 3′ genomic region from Ceriporiopsissubvermispora genomic DNA. Primer 995376:5′-GGTCGTCTCCCGCGAGGAATTCATGCACTC (SEQ ID NO: 9) G-3′ Primer 995377:5′-CTAGACAACTTCGAACGCCTTGCCA-3′ (SEQ ID NO: 10)

The 3′ fragment was amplified by PCR using the Expand High Fidelity PCRsystem (Roche Diagnostics, Mannheim, Germany). The PCR amplificationreaction mixture contained 4 μl of Ceriporiopsis subvermispora genomicDNA, 1 μl of primer 995376 (50 pmol/μl), 1 μl of primer 995377 (50pmol/μl), 5 μl of 10×PCR buffer (Roche Diagnostics, Mannheim, Germany)with 15 mM MgCl₂, 1 μl of dNTP mix (10 mM each), 37.25 μl of water, and0.75 μl of DNA polymerase mix (3.5 U/μl, Roche Diagnostics, Mannheim,Germany). The reaction was incubated in a Eppendorf Mastercyclerthermocycler programmed for 1 cycle at 94° C. for 2 minutes; 10 cycleseach at 94° C. for 15 seconds, 55° C. for 30 seconds, and 72° C. for 1.5minutes; 15 cycles each at 94° C. for 15 seconds, 55° C. for 30 seconds,and 72° C. for 1.5 minutes plus 5 second elongation at each successivecycle; 1 cycle at 72° C. for 7 minutes; and a 4° C. hold.

A re-amplication of the above described reaction was performed using 4μl of the reaction mix, 1 μl of primer 995376 (50 pmol/μl), 1 μl ofprimer 995377 (50 pmol/μl), 5 μl of 10×PCR buffer with 15 mM MgCl₂, 1 μlof dNTP mix (10 mM each), 37.25 μl of water, and 0.75 μl of DNApolymerase mix (3.5 U/μl). The reaction was incubated in a EppendorfMastercycler thermocycler programmed for 1 cycle at 94° C. for 2minutes; 10 cycles each at 94° C. for 15 seconds, 55° C. for 30 seconds,and 72° C. for 1.5 minutes; 15 cycles each at 94° C. for 15 seconds, 55°C. for 30 seconds, and 72° C. for 1.5 minutes plus 5 second elongationat each successive cycle; 1 cycle at 72° C. for 7 minutes; and 4° C.hold.

Ten microliters of the PCR reaction was mixed with 1 μl of 10×DNAloading dye and run on a 1.0% (w/v) agarose gel using TAE buffer. Theproducts were observed with UV light on a Nucleotech gel visualizationsystem. The PCR product (750 bp) was directly ligated into thepPCR2.1-TOPO vector (Invitrogen, Carlsbad, Calif.) according to themanufacturer's instructions. A 1 μl volume of fresh PCR product, 3 μl ofwater, and 1 μl of the TOPO cloning vector were mixed with a pipette andincubated on the bench top for 5 minutes.

After the incubation, 2 μl of the mixture was used to transform OneShotcompetent E. coli cells. A 2 μl volume of the ligation mixture was addedto the E. coli and incubated on ice for 5 minutes. Subsequently, thecells were heat shocked for 30 seconds at 42° C., and then placed on icefor 2 minutes. A 250 μl volume of SOC medium was added to the cells andthe mixture was incubated for 1 hour at 37° C. and 250 rpm. After theincubation the colonies were spread on 2×YT plates supplemented with 100μg of ampicillin per ml and incubated at 37° C. overnight for selectionof the plasmid.

Six colonies that grew on the plates were picked with a steriletoothpick and grown overnight at 37° C., 250 rpm in a 15 ml Falcon tubecontaining 3 ml of Luria Broth (LB) and 100 μg/ml of ampicillin. Theplasmids were isolated using the QIAGEN robot protocol described inExample 5. A 4 μl volume of the resulting plasmid minipreps was digestedwith 0.5 μl of 10 U/μl EcoRI, 2 μl of 10×SURE/Cut buffer H (RocheDiagnostics, Mannheim, Germany, and 13.5 μl water for 2 hours at 37° C.The digestion reactions were analyzed by agarose gel electrophoresis aspreviously described for the PCR reaction. The resulting plasmidcontaining 3′ genomic sequence of the Ceriporiopsis subvermisporaoxaloactetate hydrolase gene was designated pBM113a.

Plasmid, pBM113a, was sequenced using 1 μl of plasmid template, 1.6 ngof M13 primer (forward or reverse), and water to 6 μl. DNA sequencingwas performed with an Applied Biosystems Model 377 Sequencer XL usingdye-terminator chemistry. DNA sequencing revealed two base pairsubstitutions in the oligomer sequence of pBM113a. Correction of the twobase pair substitution within the pBM113a oligo sequence wasaccomplished using a QuikChange™ Site-Directed Mutagenesis Kit(Stratagene Cloning Systems, La Jolla, Calif.) according to themanufacturer's instructions with the following pairs of mutagenesisprimers: Primer 995424: 5′-CTGATAGCCGGCGCAATCTTCTCTCTCGAGT (SEQ ID NO:11) CTTGCGTCGCG-3′ Primer 995425: 5′-CGCGACGCAAGACTCGAGAGAGAAGATTGCG(SEQ ID NO: 12) CCGGCTATCAG-3′

The PCR amplification reaction mixture contained 1 μl of pBM113a, 125 ngof primer 995424, 125 ng of primer 995425, 5 μl of 10×PCR buffer, 1 μlof dNTP mix (10 mM each), 40 μl of water, and 1 μl of pfu Turbo DNApolymerase (2.5 u/μl). The reaction was incubated in a EppendorfMastercycler thermocycler programmed for 1 cycle at 95° C. for 30seconds; and 14 cycles each at 95° C. for 30 seconds, 68° C. for 9minutes. After the completion of the PCR reaction, 1 μl of DpnI wasadded to the mixture and allowed to digest at 37° C. for 1 hour.Following digestion, the reaction mixture was added to transformSolopack Gold Supercompetent cells (Staratgene, La Jolla, Calif.). Oneμl of the mixture was added to cells thawed on ice. The cell/DNA mixturewas incubated on ice for 30 minutes, followed by a 60 second heat pulsein a 54° C. water bath. A 250 μl volume of SOC medium was added to thecells and the mixture was incubated for 1 hour at 37° C. and 250 rpm.After the incubation colonies were spread on 2×YT plates supplementedwith 100 μg of ampicillin per ml and incubated at 37° C. overnight forselection of the plasmid. Four colonies that grew on the plates werepicked with a sterile toothpick and grown overnight at 37° C., 250 rpmin a 15 ml Falcon tube containing 3 ml of Luria Broth (LB) and 100 μg/mlof ampicillin. The plasmids were isolated using the QIAGEN robotprotocol described in Example 5. A resulting plasmid, pBM114a, wassequenced using 1 μl of plasmid template, 1.6 ng of M13 primer (forwardor reverse), and water to 6 μl. DNA sequencing was performed with anApplied Biosystems Model 377 Sequencer XL using dye-terminatorchemistry. From the sequence information, the 3′ genomic sequence of theCeriporiopsis subvermispora oxaloactetate hydrolase gene was deduced.

Example 11 Construction of Full-Length Ceriporiopsis SubvermisporaOxaloactetate Hydrolase Genomic Clone

Plasmid pBM115a (FIG. 2), containing the full-length oxaloacetatehydrolase genomic sequence was constructed. Plasmid, pBM114a, wasdigested with SmaI and BamHII, purified by agarose gel electrophoresisusing standard methods (Sambrook et al., 1989, supra), and ligated topCsubHP4 previously digested with BamHI and SmaI and then treated withshrimp alkaline phosphatase (Roche Diagnostics, Mannheim, Germany)following the manufacturer's protocols.

E. coli cells containing pBM115a were deposited with the AgriculturalResearch Service Patent Culture Collection, Northern Regional ResearchCenter, 1815 University Street, Peoria, Ill., 61604, as NRRL B-30669,with a deposit date of Jun. 11, 2003.

The nucleotide sequence (SEQ ID NO: 1) and deduced amino acid sequence(SEQ ID NO: 2) of the Ceriporiopsis subvermispora oxaloacetate hydrolaseare shown in FIG. 1. The insert contains an open reading frame of 1418kb encoding a polypeptide of 363 amino acids. The open reading frame isinterrupted by 6 introns of 58, 53, 55, 51, 49, and 60 bp. The % G+Ccontent is 58.3%. The oxaloacetate hydrolase has a predicted molecularweight of 38261 kDa.

A comparative alignment of oxaloacetate hydrolase sequences wasdetermined using the Clustal W method (Higgins, 1989, CABIOS 5: 151-153)using the LASERGENE™ MEGALIGN™ software (DNASTAR, Inc., Madison, Wis.)with an identity table and the following multiple alignment parameters:Gap penalty of 10 and gap length penalty of 10. Pairwise alignmentparameters were Ktuple=1, gap penalty=3, windows=5, and diagonals=5. Thealignment showed that the deduced amino acid sequence of theCeriporiopsis subvermispora oxaloacetate hydrolase gene shared 40.8%identity to the deduced amino acid sequence of an Aspergillus nigeroxaloacetate hydrolase (GenSeq aay95923).

Deposit of Biological Material

The following biological material has been deposited under the terms ofthe Budapest Treaty with the Agricultural Research Service PatentCulture Collection, Northern Regional Research Center, 1815 UniversityStreet, Peoria, Ill., 61604, and given the following accession number:Deposit Accession Number Date of Deposit E. coli pBM115a NRRL B-30669Jun. 11, 2003

The strain has been deposited under conditions that assure that accessto the culture will be available during the pendency of this patentapplication to one determined by the Commissioner of Patents andTrademarks to be entitled thereto under 37 C.F.R. §1.14 and 35 U.S.C.§122. The deposit represents a substantially pure culture of thedeposited strain. The deposit is available as required by foreign patentlaws in countries wherein counterparts of the subject application, orits progeny are filed. However, it should be understood that theavailability of a deposit does not constitute a license to practice thesubject invention in derogation of patent rights granted by governmentalaction.

The invention described and claimed herein is not to be limited in scopeby the specific aspects herein disclosed, since these aspects areintended as illustrations of several aspects of the invention. Anyequivalent aspects are intended to be within the scope of thisinvention. Indeed, various modifications of the invention in addition tothose shown and described herein will become apparent to those skilledin the art from the foregoing description. Such modifications are alsointended to fall within the scope of the appended claims. In the case ofconflict, the present disclosure including definitions will control.

Various references are cited herein, the disclosures of which areincorporated by reference in their entireties.

1. An isolated polypeptide having oxaloacetate hydrolase activity,selected from the group consisting of: (a) a polypeptide having an aminoacid sequence which has at least 70% identity with SEQ ID NO: 2; (b) apolypeptide which is encoded by a polynucleotide which hybridizes underat least medium stringency conditions with (i) SEQ ID NO: 1, (ii) thecDNA sequence contained in SEQ ID NO: 1, or (iii) a complementary strandof (i) or (ii); and (c) a variant comprising a conservativesubstitution, deletion, and/or insertion of one or more amino acids ofSEQ ID NO:
 2. 2. The polypeptide of claim 1, having an amino acidsequence which has at least 70% identity with SEQ ID NO:
 2. 3.(Canceled).
 4. (Canceled).
 5. (Canceled).
 6. (Canceled).
 7. (Canceled).8. The polypeptide of claim 1, comprising the amino acid sequence of SEQID NO:
 2. 9. The polypeptide of claim 1, which consists of SEQ ID NO: 2or a fragment thereof having oxaloacetate hydrolase activity.
 10. Thepolypeptide of claim 9, which consists of SEQ ID NO:
 2. 11. Thepolypeptide of claim 9, which consists of SEQ ID NO:
 2. 12. Thepolypeptide of claim 1, which is encoded by a polynucleotide whichhybridizes under medium stringency conditions-with (i) SEQ ID NO: 1,(ii) the cDNA sequence contained in SEQ ID NO: 1, or (iii) acomplementary strand of (i) or (ii).
 13. (Canceled).
 14. (Canceled). 15.The polypeptide of claim 1, wherein the polypeptide is a variantcomprising a conservative substitution, deletion, and/or insertion ofone or more amino acids of SEQ ID NO:
 2. 16. The polypeptide of claim 1,which is encoded by the polynucleotide contained in plasmid pBM115awhich is contained in E. coli NRRL B-30669.
 17. An isolatedpolynucleotide comprising a nucleotide sequence which encodes thepolypeptide of claim
 1. 18. (Canceled).
 19. A nucleic acid constructcomprising the polynucleotide of claim 17 operably linked to one or morecontrol sequences that direct the production of the polypeptide in anexpression host.
 20. (Canceled).
 21. A recombinant host cell comprisingthe nucleic acid construct of claim
 19. 22. A method for producing thepolypeptide of claim 1 comprising (a) cultivating a cell, which in itswild-type form is capable of producing the polypeptide, under conditionsconducive for production of the polypeptide; and (b) recovering thepolypeptide.
 23. A method for producing the polypeptide of claim 1comprising (a) cultivating a host cell comprising a nucleic acidconstruct comprising a nucleotide sequence encoding the polypeptideunder conditions conducive for production of the polypeptide; and (b)recovering the polypeptide.
 24. A method for producing a mutant of aparent cell, which comprises disrupting or deleting a nucleotidesequence encoding the polypeptide of claim 1, which results in themutant producing less of the polypeptide than the parent cell.
 25. Amutant cell produced by the method of claim
 24. 26. (Canceled). 27.(Canceled).
 28. An isolated polynucleotide obtained by (a) hybridizing apopulation of DNA under medium stringency conditions with (i) SEQ ID NO:1, (ii) the cDNA sequence contained in SEQ ID NO: 1, or (iii) acomplementary strand of (i) or (ii); and (b) isolating the hybridizingpolynucleotide, which encodes a polypeptide having oxaloacetatehydrolase activity.
 29. (Canceled).
 30. (Canceled).
 31. (Canceled). 32.(Canceled).
 33. (Canceled).
 34. A method for producing the polypeptideof claim 1, comprising (a) cultivating a transgenic plant or a plantcell comprising a polynucleotide encoding the polypeptide underconditions conducive for production of the polypeptide; and (b)recovering the polypeptide.
 35. A transgenic plant, plant part or plantcell, which has been transformed with a polynucleotide encoding thepolypeptide of claim
 1. 36. A method of delignifying a materialcomprising (a) treating the material with a microorganism comprising thenucleic acid construct of claim 19, and (b) recovering the treatedmaterial.
 37. (Canceled).
 38. (Canceled).
 39. (Canceled).